EP3158067B1

EP3158067B1 - Compositions and methods for regulating gene expression via rna interference

Info

Publication number: EP3158067B1
Application number: EP15812530.2A
Authority: EP
Inventors: Alberto Iandolino; Juan Pedro SANCHEZ
Original assignee: Monsanto Technology LLC
Current assignee: Monsanto Technology LLC
Priority date: 2014-06-23
Filing date: 2015-06-22
Publication date: 2020-08-12
Anticipated expiration: 2035-06-22
Also published as: CN106795515A; CA2953347A1; US10988764B2; WO2015200223A1; AU2015280252A1; US20160160212A1; CN106795515B; UY36189A; EP3158067A1; EP3158067A4; AR100946A1

Description

FIELD

The present disclosure provides compositions and methods for regulating gene expression via RNA-mediated silencing. The present disclosure also provides compositions and methods to optimize the directional processing of a dsRNA molecule into small RNA (sRNA) duplexes.

BACKGROUND

Non-transgenically produced, exogenous nucleic acid molecules, for example, double stranded RNA (dsRNA) molecules, have been shown to trigger the silencing of a plant endogenous gene after being applied topically to a plant leaf or by soaking a seed with a solution containing the nucleic acid molecules. See U.S. Patent Publication Nos. 2011/0296556 , and 2013/0318657 . Therefore, plant traits can be modified by introducing into a plant or seed dsRNA molecules that specifically regulate the expression of genes responsible for those traits.
RNA-mediated sequence-specific gene regulation, also called RNA interference (RNAi), starts with a dsRNA that comprises a RNA strand that complements the sequence of a gene of interest. The dsRNA molecule is then processed into shorter fragments of approximately 21-24 nucleotides by an RNase Ill-related enzyme (Dicer). These fragments, called small interfering RNAs (siRNAs), get incorporated into the RNA-induced silencing complex (RISC). After additional processing, the siRNAs are transformed into single-stranded RNAs that act as guide sequences to recognize and direct the cleavage of target gene transcripts.
Plant cells can produce dsRNAs. A tomato RNA-Dependent RNA Polymerase (RDR) has been reported to produce dsRNAs with 1-nt or 2-nt 3 '-overhangs at both termini. See Schiebel et al., J. Biol. Chem. 263: 11858-67 (1993); Rajeswaran et al., Nucleic Acid Res., 40(13):6241-54 (2012). It is desired to have a dsRNA processed into siRNAs, and eventually single-stranded RNAs, following a predictable and pre-programmed pattern. The instant application provides and discloses, among others, sequence and structural features that are incorporated into a dsRNA molecule to improve the predictability of the processing of a dsRNA into siRNAs functional in guiding the silencing of intended target genes. Therefore, the instant application provides nucleic acid molecules with higher efficacy in promoting gene regulation and trait modification.
Chang et al. (2009 Mol. ) describe dual -target gene silencing by using long, synthetic siRNA duplexes without triggering antiviral response. WO2010/093788 discloses that compound DsiRNA agents can be generated using site-specific RNase H-cleavable double stranded nucleic acid double stranded nucleic acid regions to attach, e.g., one DsiRNA moiety to another DsiRNA moiety and/or one DsiRNA moiety to a functional group and/or payload. Because such double stranded nucleic acid joining sequences are site-specifically RNase H-cleavable, the bifunctional molecule is cleaved into DsiRNAs which bear terminal ends that orient dicer cleavage.

SUMMARY

The instant disclosure provides compositions and methods for regulating gene expression. In one aspect, the instant disclosure provides exogenous trigger molecules for RNAi-mediated silencing in plants.
In one aspect, the instant disclosure provides a double-stranded RNA (dsRNA) molecule comprising a). a first strand comprising in the 5' to 3' direction i) a first sequence that is identical to at least 18 consecutive nucleotides of a first target nucleotide sequence; and ii) a second sequence that is identical to at least 18 consecutive nucleotides of a second target nucleotide sequence; and b).a second strand comprising in the 5' to 3' direction, a 5'-overhang, a nucleotide sequence that is complementary to the first strand, and a 2 nucleotide 3 '-overhang, wherein the 5 '-overhang is at least 5 nucleotides in length.
In another aspect, the instant disclosure provides a dsRNA molecule comprising: a). a first strand comprising in the 5' to 3' direction, i). a first nucleotide sequence that is identical to at least 18 consecutive nucleotides of a first target nucleotide sequence; ii). a second nucleotide sequence comprising 2 or more As; and iii). a third nucleotide sequence that is identical to at least 18 consecutive nucleotides of a second targeted nucleotide sequence or at least 18 consecutive nucleotides of the first target nucleotide sequence; and b). a second strand comprising in the 5' to 3' direction, a 5 nucleotide 5'-overhang, a nucleotide sequence that is complementary to the first strand, and a 2 nucleotide 3'-overhang. In some embodiments, the first and second target nucleotide sequences are identical.
In a further aspect, the instant disclosure also provides a composition comprising a dsRNA molecule disclosed herein. In another aspect, the instant disclosure provides a plant, plant part, or seed comprising a dsRNA molecule disclosed herein, wherein the dsRNA molecule is exogenous to the plant, plant part, or seed.
In one aspect, the instant disclosure provides a method of regulating expression of at least one target gene, comprising applying onto the surface of a plant or plant part a composition comprising a dsRNA molecule disclosed herein, wherein the dsRNA molecule comprises a first strand comprising in the 5' to 3' direction i) a first sequence that is identical to at least 18 consecutive nucleotides of the first target nucleotide sequence and ii) a second sequence that is identical to at least 18 consecutive nucleotides of a second target nucleotide sequence; and b) a second strand that is complementary to the first strand, and a 2 nucleotide 3 '-overhang, wherein the 5' overhang is at least 5 nucleotides in length..
In another aspect, the instant disclosure also provides a method of improving the efficiency of a dsRNA molecule in producing desired small RNAs in a plant, plant part or seed, comprising providing to the plant, plant part or seed a dsRNA molecule disclosed herein, wherein the production of the 21-24 nucleotide small RNAs is directionally biased towards the 3' end of the second strand of the dsRNA molecule.
In one aspect, the instant disclosure provides a dsRNA molecule, wherein the processing of a population of the dsRNA molecules into one or more 21-24mer small RNAs (sRNAs) preferentially starts from an end having a 3' overhang, and wherein at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of detectable 21-24mer sRNAs processed therefrom comprise a sequence identical to a sequence of a first double-stranded portion of at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides immediately adjacent to the 3' overhang.
In one aspect, the instant disclosure provides a dsRNA molecule, wherein the first cleavage of the dsRNA molecule by a Dicer-like protein is at a position of about 21 to 24 nucleotides from the 3' terminus of a 3' overhang, and wherein at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of detectable 21-24mer sRNAs processed from the population of the dsRNA molecules comprise a sequence identical to a sequence of a first double-stranded portion of at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides immediately adjacent to the 3' overhang.
In one aspect, the instant disclosure provides a dsRNA molecule, wherein the processing of a population of the dsRNA molecule into one or more 21-24mer sRNAs preferentially starts from one end of the dsRNA molecule, and wherein the most abundant detectable 21-24mer sRNAs processed from the population of the dsRNA molecules comprise a sequence identical to a sequence of a first double-stranded portion of at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides immediately adjacent to the 3' overhang. In another aspect, the second most abundant detectable 21-24mer sRNAs processed from a population of dsRNA molecules as disclosed herein comprise a sequence identical to a sequence immediately adjacent to the first double-stranded portion.
In one aspect, the instant disclosure provides a dsRNA molecule comprising a 5' overhang at a first end of the dsRNA molecule, wherein the processing of the dsRNA molecule into one or more sRNAs preferentially starts from a second end of the dsRNA molecule, and wherein the first and second ends are opposite ends of the dsRNA molecule.
In one aspect, a dsRNA molecule or directional trigger of the instant disclosure comprises a first end portion comprising a 3' overhang, a second end portion comprising a 5' overhang, and two or more target-specific sequences that are adjoined by one or more linker sequences.
In one aspect, the instant disclosure provides a directional trigger comprising an exogenous dsRNA molecule having a preferential directionality when processed into sRNAs by a Dicer-like protein. In one aspect, a directional trigger of the instant disclosure comprises a 3' overhang. In some embodiments, the 3' overhang is at least 2 nucleotides in length. In some embodiments, the 3' overhang has the sequence UA, UU, AA, AU, UG or UC. In another aspect, a directional trigger of the instant disclosure comprises a 5' overhang. In some embodiments, the 5' overhang has a high GC content. In some embodiments, the 5' overhang is, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides in length. In some embodiments, the 5' overhang is at least 5 nucleotides in length. In a further aspect, a directional trigger has a 3' overhang and a 5' overhang on the same strand. In a further aspect, a directional trigger has a 3' overhang and a 5' overhang on the antisense strand. In a further aspect, a directional trigger has a 5'G on the sense strand.
In a further aspect, the instant disclosure provides a plant, plant part or seed comprising a dsRNA molecule disclosed herein, wherein the dsRNA molecule is exogenous to the plant, plant part or seed.
In one aspect, a dsRNA molecule or directional trigger as disclosed herein has a pre-programmed processing pattern for generating sRNAs where sRNA processing starts from an end comprising a 3' overhang and continues in a phased manner with about a 21-nucleotide phase. In a further aspect, a directional trigger is capable of producing one or more sRNA duplexes that have strand-selectivity by preferentially having their antisense strands as guide strands. In some aspect, at least one sRNA duplex produced by a directional trigger as disclosed herein comprises a Uracil or a Uracil-Uracil di-nucleotide at the 5' end of its antisense strand.
In one aspect, the dsRNA molecule or directional trigger disclosed herein is a chimera comprising two or more target-specific sequences that, when the directional trigger is cleaved by a Dicer-like protein, yield the same number of sRNAs, each of which has one target-specific sequence. In one aspect, two or more target-specific sequences are immediately adjacent to each other in a directional trigger. In one aspect, two or more target-specific sequences are not adjacent in a directional trigger. In one aspect, two or more target-specific sequences are not contiguous in a directional trigger. In one aspect, two or more target-specific sequences of a directional trigger are from two or more different genes. In another aspect, two or more target-specific sequences in a directional trigger are derived from a same gene but non-contiguous in that gene. In a further aspect, two or more target-specific sequences of a directional trigger has essentially identical sequences. In another aspect, a directional trigger further comprises one or more AU-rich linker sequences adjoining two or more target-specific sequences.
In one aspect, at least 50% of sRNAs processed from a directional trigger as disclosed herein are from the 3' end of the antisense strand of the directional trigger. In another aspect, at least 50% of sRNAs processed from a directional trigger as disclosed herein comprise a sequence from the 3' end of the antisense strand of the directional trigger.
In one aspect, a directional trigger as disclosed herein is not from a viral vector. In another aspect, a directional trigger as disclosed herein is not produced from a natural viral infection.
In one aspect, a directional trigger as disclosed herein is chemically synthesized or enzymatically produced. In another aspect, a directional trigger as disclosed herein is chemically modified. In one aspect, chemical modification of a directional trigger is capable of enhancing delivery of the molecule into a plant cell or stability of the molecule in a plant cell. In another aspect, chemical modification of a directional trigger is selected from the group consisting of a cholesterol moiety and a modified nucleotide.
In one aspect, a directional trigger as disclosed herein is capable of regulating gene expression via a mechanism selected from the group consisting of RNA cleavage, translation or transcription attenuation, and DNA modification.
In one aspect, a directional trigger as disclosed herein comprises two or more target-specific sequences from one or more target genes selected from the group consisting of an endogenous plant gene, a transgene, an essential gene of a plant pest or pathogen, a plant gene providing resistance to a herbicide, and a plant gene involved in abiotic or biotic stress tolerance.
In one aspect, the instant disclosure provides a double-stranded RNA (dsRNA) molecule comprising a) two or more sRNA trigger sequences that encode the same number of sRNAs, wherein the two or more sRNA trigger sequences are not found in a single naturally occurring molecule or not contiguous in a single naturally occurring molecule, b) a length between about 45 and about 75 nucleotides, c) one or more Adenine- or Uracil-rich linker sequences adjoining the two or more sRNA trigger sequences, d) a 3' overhang in the antisense strand of the dsRNA molecule, e) a Uracil at positions 20, 21 in the antisense strand, the positions 20 and 21 are the 20th and 21 st nucleotides relative to terminus of the 3' overhang, respectively, and f) a 5' overhang of 5 nucleotides.
In one aspect, the instant disclosure provides a composition comprising a directional trigger as disclosed herein, and a transferring agent which facilitates transfer of the directional trigger from the surface of a plant into a cell of a plant. In one aspect, a composition as disclosed herein comprises a transferring agent selected from the group consisting of a surfactant and a salt. In one aspect, a transferring agent of the instant disclosure comprises a humectant or a chelating agent.
In one aspect, a composition as disclosed herein comprises a directional trigger and an organosilicone surfactant. In another aspect, a composition as disclosed herein comprises a directional trigger and a silicone polyether copolymer. In one aspect, a composition as disclosed herein comprises a directional trigger and an organic or an inorganic salt.
In a further aspect, the instant disclosure provides a plant or seed treated with a composition comprising a directional trigger as disclosed herein, and a transferring agent. In another aspect, the instant disclosure provides a plant, a plant part, or seed comprising a directional trigger as disclosed herein.
In a further aspect, the instant disclosure provides a method of applying a directional trigger, or a composition made thereof, to a plant, plant part or seed to confer a beneficial trait.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C : Without being limited by any particular theory, overhangs on dsRNA molecules influence both the processing of the molecule and the molecule's ability to promote gene silencing. FIG. 1A illustrates six dsRNA triggers each of which comprises two strands of ∼50 nucleotides with overhangs of various lengths. These dsRNA triggers target a tomato (Solanum lycopersicum, Sl) 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. Trigger 1 has blunt ends. Trigger 2 has 2-base 3' overhangs on both ends. Triggers 3 to 6 have on each end 2-base, 5-base, 10-base and 15-base 5'overhangs, respectively. All triggers are 5'-end-labeled with P32. FIG. 1B illustrates a denaturing polyacrylamide gel analysis of the processing of triggers 1 to 6 after a 2-hr incubation in wheat germ extract (WGE). The presence of 5' overhangs delays or prevents the processing of a dsRNA trigger molecule. The arrowhead denotes the presence of siRNAs, while the arrow indicates full-length trigger molecules. A size ladder is marked to the left of the image. FIG. 1C shows Taqman quantitative PCR data illustrating various extents of down-regulation of a EPSPS gene in Nicotiana benthamiana protoplasts by dsRNA triggers 1 to 6. The degree of target silencing by trigger molecules having 5' overhangs on both ends decreases as the length of the 5' overhangs increases.
Figures 2A-2B : A comparison between a dsRNA molecule synthesized by a RNA dependent RNA polymerase (RDR, RDR6 shown as an example) ( FIG. 2A ), and one embodiment of a directional trigger as disclosed herein which comprises on their antisense strands a 2-nt 3' overhang ("3' initiator overhang") and a 5' overhang ("5' blocker overhang") ( FIG. 2B ). In contrast to an RDR-synthesized dsRNA molecule that has 3' overhangs at both ends and lacks directionality when processed by a Dicer protein, a directional trigger is processed predominantly starting for the end with a 3' initiator overhang.
Figure 3 : A schematic representation of the rational design and processing of a directional dsRNA trigger, which is preferentially processed by Dicer-like proteins into two distinct, functional siRNAs in a phased manner. Design concepts include, without limitations, directional initiation of processing by Dicer-like proteins from one end with a 3' overhang, and use of a 5' overhang in the opposite end blocking initiation of processing by Dicer-like proteins from that end. A rationally designed directional chimeric dsRNA trigger molecule predominantly produces siRNA products that are in a proper phase to target two genes of interest (GOI1 and GOI2). Meanwhile, the processing of a directional dsRNA trigger leads to only a minor fraction of its siRNA products with an aberrant phase. "Aberrant" phased siRNA products have at best a partial complementarity with a target sequence which are not capable of triggering silencing of the target.
Figure 4 : A schematic representation of a directional dsRNA trigger (52 nucleotides shown for illustration purposes) which is preferentially processed by a Dicer-like protein to at least two distinct siRNAs in a phased manner. As shown, the two distinct siRNAs recognize sequences in the same or different target genes (GOI1 only, or GOI1 and GOI2). Both siRNA1 and siRNA2 comprise a UU di-nucleotide at the 5' end of the antisense strand and a G at the 5' end of the sense strand. The antisense strands of siRNA1 and siRNA2 starting with a UU di-nucleotide are preferentially loaded into Argonaute proteins (AGO) and recognize target gene mRNA molecules leading to target gene silencing.
Figures 5A-5B : A schematic comparison between one embodiment of a directional dsRNA trigger and a non-directional dsRNA trigger. The schematic drawings and the following explanation are provided for illustration purposes only, and are not bound to any scientific theory or mechanism. In FIG. 5A , the sense strand of a directional trigger comprises target-specific sequences from genes of interest (GOIs). Target- specific sequences 1 and 2 as shown in the figure can be from a same GOI or different GOIs. The antisense strand of a directional trigger comprises both a 3' overhang (2-nt exemplified) and a 5' overhang. A Dicer-like protein cleaves a first 21-24mer (siRNA1) from a directional trigger preferentially starting from the end with a 3' overhang, and also produces a second 21-24mer (siRNA2) which is immediately next to the first 21-24mer (e.g., in phase with the first 21-24mer). Accordingly, a Directional dsRNA trigger produces a group of 21-24mers (two 21-24mers are shown in the figure) in a phased manner with siRNA1 and siRNA2 being the predominant species. Both siRNA1 and siRNA2 comprise a UU di-nucleotide at the 5' end of the antisense strand and a G at the 5' end of the sense strand. Antisense strands of siRNA1 and siRNA2 starting with a UU di-nucleotide are preferentially loaded into Argonaute proteins (AGO), and are also called guide strands that guide the recognition of target gene mRNA sequences and lead to target gene silencing.
A non-directional dsRNA trigger can be chimeric or non-chimeric, blunt-ended, having 3' overhangs on both ends, or a combination of these features. Shown in FIG. 5B , a chimeric trigger has two 3' overhangs. A non-directional dsRNA trigger has no directionality bias towards either end of the trigger when processed by a dicer-like protein. Accordingly, 21-24mers produced from a non-directional dsRNA trigger are more heterogeneous. In-phase 21-24mers (e.g., siRNA1' and siRNA2') represent only a fraction of the total pool of 21-24mers which also comprise substantial out-of-phase 21-24mers (e.g., siRNA3' and siRNA4'). As such, a non-directional dsRNA trigger produces more diluted in-phase 21-24mers compared to a directional trigger.
Further, 21-24mers produced from a non-directional dsRNA trigger lack a UU di-nucleotide at the 5' end of their antisense strand and a G at the 5' end of their sense strand. Accordingly, neither the antisense strand, nor the sense strand is preferentially loaded into a AGO protein. Instead, each strand of every 21-24mer can potentially be loaded into an AGO protein as a guide strand. Guide strands 1' and 2' are complementary to target sequences and capable of recognizing target molecules to cause silencing. Therefore, a non-directional dsRNA trigger produces more diluted guide strands that are effective in causing silencing compared to guide strands produced from a directional trigger.
Figures 6A-6D : Directional triggers against two Arabidopsis target genes generate two functional siRNAs against their intended targets in Arabidopsis protoplasts. FIG. 6A illustrates schematic representations of three directional chimeric dsRNA triggers tested. These directional dsRNA triggers each comprise two target sequences, each of which is from AtEPSPS or AtCUT1 (SEQ ID NO:7/SEQ ID NO:58), or both of which are from either AtEPSPS (SEQ ID NO:8/SEQ ID NO:59) or AtCUT1 (SEQ ID NO:9/SEQ ID NO:60). FIG. 6B shows activity of chimeric triggers in wheat germ extract (WGE) silencing a fusion target mRNA AtEPSPS1:Fluc. AtEPSPS1:Fluc is an mRNA fusion between a full-length luciferase coding sequence and an AtEPSPSl coding sequence which is targeted by triggers SEQ ID NO:7/SEQ ID NO:58, SEQ ID NO:8/SEQ ID NO:59, and SEQ ID NO:10/SEQ ID NO:61. SEQ ID NO:10/SEQ ID NO:61 is a non-directional dsRNA trigger. FIG. 6C shows activity of chimeric triggers in wheat germ extract (WGE) silencing a target mRNA. AtCUT1:Fluc is an mRNA fusion between a full-length luciferase coding sequence and an AtCUT1 coding sequence that is targeted by triggers SEQ ID NO:7/SEQ ID NO:58 and SEQ ID NO:9/SEQ ID NO:60, but not SEQ ID NO:8/SEQ ID NO:59. The y-axis reflects relative luciferase activity normalized to Renilla luciferase. Error bars represent standard deviations. For WGE incubation, fusion target mRNA were used at 3 pmol/µl, while triggers were used at 60 pmol/µl. FIG. 6D shows q-PCR results for AtEPSPSl expression levels ("EPSPS RQ") in Arabidopsis protoplasts after treatment with directional dsRNA triggers from FIG. 5A . Directional triggers containing at least one siRNA sequence against AtEPSPS1 (SEQ ID NO:7/SEQ ID NO:58 and SEQ ID NO:8/SEQ ID NO:59) are capable of specifically downregulating AtEPSPSl expression, while directional trigger against AtCUT1 (SEQ ID NO:9/SEQ ID NO:60) cannot silence AtEPSPSl. The percentages of AtEPSPSl down-regulation in each treatment compared to the no-trigger control are shown in the figure. All triggers were tested at a 200 pmol dosage except trigger SEQ ID NO:8/SEQ ID NO:59 which contains two distinct siRNAs both targeting AtEPSPS1 and was evaluated at a 50 pmol dosage. This lower dosage of SEQ ID NO:8/SEQ ID NO:59 achieving target down-regulation close to that by a higher concentration of SEQ ID NO:7/SEQ ID NO:58 shows that SEQ ID NO:8/SEQ ID NO:59 having two target-specific sequences from AtEPSPS1 has a higher efficacy compared to SEQ ID NO:7/SEQ ID NO:58.
Figures 7A-7E : Directional chimeric dsRNA triggers targeting tomato (Solanum lycopersicum, Sl) phytoene desaturase (PDS) and EPSPS genes are effective in triggering silencing in Nicotiana benthamiana (Nb) protoplasts. FIG. 7A illustrates schematic representations of three directional triggers tested. SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63 each comprise two target sequences, one from SlPDS and the other from SlEPSPS. Arrangements of the two target sequences are reversed between SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63. FIG. 7B shows activity of chimeric triggers in wheat germ extract (WGE) silencing a target mRNA SlPDS:Fluc. SlPDS: Fluc is an mRNA fusion between a full-length luciferase coding sequence and a SlPDS coding sequence which is targeted by triggers SEQ ID NO: 11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63, but not SEQ ID NO:9/SEQ ID NO:60. FIG. 7C shows activity of chimeric triggers in WGE silencing a target mRNA SlEPSPS:Fluc. SlEPSPS:Fluc is an mRNA fusion between a full-length luciferase coding sequence and a SlEPSPS coding sequence which is targeted by triggers SEQ ID NO: 11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63, but not SEQ ID NO:9/SEQ ID NO:60. The y-axis reflects relative luciferase activity normalized to an internal Renilla luciferase control reporter. Error bars represent standard deviations. For WGE incubation, target mRNAs were used at 3 pmol/µl, while each trigger was used at 60 pmol/µl. FIG. 7D shows Northern blot results demonstrating down-regulation of a NbEPSPS gene by triggers SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63 in Nicotiana benthamiana protoplasts and a rRNA control below. FIG. 7E lists quantification results of target gene (Gene of Interest, GOI) downregulation by triggers SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63 in Nicotiana benthamiana protoplasts via either a 5' probe or a 3' probe. %KD refers to the percentage of gene knockdown by each trigger compared to a no-trigger control.
Figure 8 : A comparison of target down-regulation in Arabidopsis protoplasts between a directional dsRNA trigger (SEQ ID NO:8/SEQ ID NO:59 targeting AtEPSPS1, see FIG. 5A ) and a non-directional trigger SEQ ID NO:10/SEQ ID NO:61 shows that a directional dsRNA trigger has a higher silencing efficacy compared to a non-directional trigger. Eight different trigger concentrations (250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, and 2.0 pmol) were tested. SEQ ID NO:14/SEQ ID NO:64 and SEQ ID NO:14/SEQ ID NO:81 represent negative control directional dsRNA triggers that do not target AtEPSPSl. Triggers are listed on the x-axis according to their concentration. For example, the first data point on the x-axis "01 250 SEQ ID NO:10/SEQ ID NO:61" refers to treatment No. 01 which uses 250 pmol of trigger SEQ ID NO:10/SEQ ID NO:61. Similarly, the data point on the x-axis "32 2.0 SEQ ID NO:14/SEQ ID NO:81" refers to treatment No. 32 which uses 2.0 pmol of trigger SEQ ID NO:14/SEQ ID NO:81. Relative q-PCR quantification of AtEPSPSl expression is shown on the y-axis. Student's t-test was performed to show statistic significance. Same quantification results are also shown in Table 2. The directional dsRNA trigger SEQ ID NO:8/SEQ ID NO:59 reduced AtEPSPS1 expression by 34% and 39% when used at 125 and 250 pmol, respectively. The non-directional dsRNA trigger SEQ ID NO:10/SEQ ID NO:61 was capable of reducing AtEPSPS1 expression by 24% at the highest dose (250 pmol), and showed no silencing activity when used at a concentration of 125 pmol or lower. When used at a same concentration (e.g., 250 pmol), directional dsRNA trigger SEQ ID NO:8/SEQ ID NO:59 is more effective in reducing AtEPSPS1 expression compared to non-directional dsRNA trigger SEQ ID NO:10/SEQ ID NO:61 (39% versus 24%).
Figures 9A-9C : A survey of siRNAs that are processed from a directional chimeric dsRNA trigger via deep sequencing demonstrates directionality. Trigger SEQ ID NO:9/SEQ ID NO:60 ( FIG. 9A ) was processed in WGE, and the collection of products were subject to deep sequencing thereafter. FIG. 9B shows size distribution for RNA products of the SEQ ID NO:9/SEQ ID NO:60 after processing in WGE on the x-axis and the number of reads of each RNA size on the y-axis. In total, 2,456,774 sequencing reads are mapped to trigger SEQ ID NO:9/SEQ ID NO:60, of which 2,107,001 reads (∼85%) are from the antisense strand of trigger SEQ ID NO:9/SEQ ID NO:60 while 349,774 sequencing reads (∼15%) are from the sense strand of trigger SEQ ID NO:9/SEQ ID NO:60. FIG. 9C shows that the size range of 21-24 (i.e., 21-24mer) contains 473,000 sequencing reads (∼19%), of which ∼90% are mapped to the 3' end of the antisense strand of trigger SEQ ID NO:9/SEQ ID NO:60 with only ∼0.4% from the 5' end of the sense strand. Analysis of the 21-24mers shows an overrepresentation of the 3' end of the antisense strand compared to the antisense 5' end (∼17% versus ∼0.4%), which supports a preferential, directional processing of the chimeric trigger starting from the end comprising a 2-nt 3' overhang. The processing patterns of two additional triggers, SEQ ID NO:7/SEQ ID NO:58 and SEQ ID NO:8/SEQ ID NO:59, into 21-24mers are shown in Table 1.
Figure 10 : A detailed analysis of the 21-24mers from the 3' end of the antisense strand processed from Trigger SEQ ID NO:9/SEQ ID NO:60 in WGE as in FIG. 7 . For each size and sequence listed, both the number (#) of sequencing reads and its percentage of the total number of 21-24mers are shown.
Figure 11 : An alignment of the most abundant putative primary siRNA duplexes (e.g., perfect-match double-stranded 21-24mers) from a 48-nt trigger sequence (SEQ ID NO:15, top row). Putative siRNA duplexes were assembled in silico from 21-24mer sequencing reads which are generated by sequencing small RNAs processed from trigger BOL5.2 in WGE. These putative siRNA duplexes are ranked based on their relative abundance estimated by the sum of their absolute frequencies. Top ranked putative duplexes (only perfect-match 21-24mers) were aligned against the BOL5.2 48-nt trigger sequence. The top 10 putative siRNA duplexes amount to ∼75 % of all perfect match reads. Among these 10 duplexes, only 3 preferentially match to the 3' side of the trigger (the dsRNA end with a 5' overhang). Four top-ranked duplexes (two 21 -nt and two 24-nt siRNAs) showed opposite strand biases. The two top-ranked 21-nt siRNAs are biased towards the antisense strand, while the two top-ranked 24-nt siRNAs have a bias to the sense strand.
Figure 12: Results of the Western Blot analysis using anti-GFP (panel A) or anti-MgChl (panel B) polyclonal antibodies. The lanes are as follows: lane I is unsilenced green tissue (under UV light) from plants treated with GFP only trigger; lane 2 is empty; lane 3 is tissue prepared from GFP silenced treatment (red spots under UV light); lane 4 is empty; lane 5 is from tissue prepared from plants treated with chimeric trigger targeting GFP/MgChl.
Figure 13: A diagram and Taqman data of the dsRNA polynucleotides tested in protoplast cells. Panel A shows the three dsRNAs being test: the directional trigger that targets both PDS and PAT1 (SEQ ID NO:68/SEQ ID NO:69) with the 5'-overhang and desired composition, the dsRNA trigger with mutations within the sequence complementary to PDS or PAT1 (SEQ ID NO:70/SEQ ID NO:71), and the blunt ended dsRNA trigger (SEQ ID NO:72/SEQ ID NO:73). Panel B shows the Taqman results obtained after RNA extraction and quantitation.

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms as used herein have the same meaning as commonly understood by one of ordinary skill in the art. One skilled in the art will recognize many methods can be used in the practice of the present disclosure. Moreover, the present disclosure is not intended to be limited by any particular scientific theory. For purposes of the present disclosure, the following terms are defined below.
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A used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.
As used herein, the term "about" indicates that a value includes the inherent variation of error for the method being employed to determine a value, or the variation that exists among experiments.
As used herein, a "dsRNA" molecule refers to a molecule comprising two antiparallel ribonucleotide strands bound together by hydrogen bonds, each strand of which comprises ribonucleotides linked by phosphodiester bonds running in the 5'-3' direction in one and in the 3'-5' direction in the other. Two antiparallel strands of a dsRNA can be perfectly complementary to each other or comprise one or more mismatches up to a degree where any one additional mismatch causes the disassociation of the two antiparallel strands. A dsRNA molecule can have perfect complementarity over the entire dsRNA molecule, or comprises only a portion of the entire molecule in a dsRNA configuration. Two antiparallel strands of a dsRNA can also be from a continuous chain of ribonucleotides linked by phosphodiester bonds, e.g., a hairpin-like structure (often also called a stem-loop structure). In some embodiments, a dsRNA molecule is identified by two SEQ ID NOs, where the first SEQ ID NO represents the sense strand of the dsRNA and the second SEQ ID NO represents the antisense strand of the dsRNA. In other embodiments, a dsRNA molecule is identified by one SEQ ID NO that represents the sense strand of the dsRNA.
As used herein, in the context of RNA-mediated gene silencing, the sense strand of a dsRNA molecule refers to a strand comprising a sequence that is identical or essentially identical to a target RNA sequence. The antisense strand of a dsRNA molecule refers to a strand having a sequence complementary to a target RNA sequence. In a DNA context, the term "antisense" refers to a nucleotide sequence that is inverted relative to its normal orientation for transcription or function and so expresses an RNA transcript that is complementary to a target gene mRNA molecule expressed within the host cell (e.g., it can hybridize to the target gene, mRNA molecule or single stranded genomic DNA through Watson-Crick base pairing) or that is complementary to a target DNA molecule such as, for example, genomic DNA present in the host cell.
As used herein, the term "overhang" refers to one or more single-stranded nucleotides at one end of a nucleic acid molecule which comprises a double stranded portion. A "3' overhang" refers to one or more single-stranded nucleotides ending in a 3' hydroxyl or modification thereof. A "3' initiator overhang" refers to a dsRNA molecule's 3' overhang of variable length, e.g., 2-nt, which favors the initiation of dsRNA processing by a Dicer-like protein from the terminus having the 3' overhang. Similarly, a "5' overhang" refers to one or more single-stranded nucleotides ending in a 5' phosphate or modifications thereof. A "5' blocker overhang" refers to a 5' overhang of a dsRNA molecule which disfavors the initiation of dsRNA processing by a Dicer-like protein from the terminus having the 5' overhang. A terminal nucleotide (or terminus) of a 3' or 5' overhang refers to the overhang's nucleotide that is furthest away from a double-stranded portion.
A frayed end refers to a double-stranded nucleic acid molecule end with a significant proportion of non-complementary sequences (e.g., nucleotides on parallel strands do not form Watson-Crick pairing).
As used herein, "small RNA (sRNA)" refers to any RNA molecule that is about 15-30 nucleotides long, preferably 21-24 nucleotides long. A "21-24mer small RNA" or "21-24mer sRNA" refers to a small RNA of 21-24 nucleotides which may be double- or single-stranded. A double-stranded 21-24mer sRNA can comprise at one or both ends one or more structures selected from the group consisting of blunt, 3' overhang, and 5' overhang. A double-stranded 21-24mer sRNA processed by a Dicer-like protein from a dsRNA precursor molecule typically comprise a 2-nt overhang at both ends.
Small RNA includes, without limitation, siRNA (small interfering RNA), miRNA (microRNA), ta-siRNA(trans activating siRNA), activating RNA (RNAa), nat-siRNA (natural anti-sense siRNA), hc-siRNA (heterochromatic siRNA), cis-acting siRNA, ImiRNA (long miRNA), IsiRNA (long siRNA) and easiRNA (epigenetically activated siRNA). Preferred sRNA molecules of the disclosure are siRNA molecules. A sRNA, in its mature form, can be either double-stranded or single-stranded, although the biogenesis of a sRNA often involves a sRNA duplex which is a double-stranded form of sRNA. While not limited by a particular theory, a sRNA duplex is often processed from a dsRNA precursor (e.g., a directional trigger as disclosed herein) by proteins, such as Dicer-like proteins.
As used herein, the term "siRNA" (also referred to herein interchangeably as "small interfering RNA"), is a class of double-stranded RNA molecules having about 18-25 nucleotides in length (e.g., 18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers). A double-stranded siRNA generally has perfect or near perfect complementarity. Without being limited by any theory, a role of siRNA is its involvement in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific target gene.
One strand of a siRNA, called "guide strand," is loaded into a RNA-induced silencing complex (RISC) and guide the recognition of a complementary mRNA molecule (target mRNA molecule) and to trigger subsequent silencing. The other strand of a siRNA, called "passenger strand," is degraded.
As used herein, the term "functional siRNA" refers to a siRNA which is effective in silencing an intended target gene.
As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression) mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding target gene.
As used herein, the phrase "immediately adjacent" refers to a position directly connected, without a gap or spacing, to a reference position or structure. Two nucleic acid sequences are immediately adjacent or contiguous when they are found in a sequence of a single molecule and lie right next to each other without any gap or spacing.
As used herein, a "synthetic sequence" refers to a nucleic acid sequence which lacks a corresponding sequence that naturally occurs.
As used herein, a "target-specific sequence" refers to a nucleic acid sequence that is essentially identical, identical, essentially complementary, or complement of any, to nucleotide sequence that occurs in a gene or gene product against which a trigger polynucleotide is directed. In this context, the term "gene" means a locatable region of genomic sequence, corresponding to a unit of inheritance, which includes regulatory regions, such as promoters, enhancers, 5' untranslated regions, intron regions, 3' untranslated regions, transcribed regions, and other functional sequence regions that may exist as native genes or transgenes in a plant genome or the genome of a pathogen. As used herein, the term "pathogen" refers to virus, viroid, bacteria, fungus, oomycetes, protozoa, phytoplasma, and parasitic plants. Depending upon the circumstances, the term target sequence or target gene can refer to the full-length nucleotide sequence of the gene or gene product targeted for suppression or the nucleotide sequence of a portion of the gene or gene product targeted for suppression. In some embodiments, a target-specific sequence can be derived from a sequence of a messenger RNA (mRNA) which, when hybridizes with a small RNA molecule and leads to the attenuation of target gene expression. In some embodiments, a target-specific sequence can be derived from a sequence of microRNAs, small interfering RNAs, and other small RNAs associated with a silencing complex (RISC) or an Argonaute protein; RNA components of ribosomes or ribozymes; small nucleolar RNAs; and other non-coding RNAs. In some embodiments, a target-specific sequence can be derived from non-translatable (non-coding) sequence, such as, but not limited to, 5' untranslated regions, promoters, enhancers, or other non-coding transcriptional regions, 3' untranslated regions, terminators, and introns. In some embodiments, a target-specific sequence can be derived from a gene encoding transcription factors, enzymes involved in the biosynthesis or catabolism of molecules of interest (such as, but not limited to, amino acids, fatty acids and other lipids, sugars and other carbohydrates, biological polymers, and secondary metabolites including alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin). Conversely, a "non-target-specific sequence" refers to any nucleic acid sequence that is not a target-specific sequence.
As used herein, the terms "trigger," "trigger polynucleotide," or "polynucleotide trigger" refers to a bioactive polynucleotide molecule that comprises a polynucleotide that substantially homologous or complementary to a polynucleotide sequence of a target gene or an RNA expressed from the target gene or a fragment thereof and functions to suppress the expression of the target gene or produce a knock-down phenotype. Trigger polynucleotides are capable of inhibiting or "silencing" the expression of a target gene. Trigger polynucleotides are generally described in relation to their "target sequence." Trigger polynucleotides may be single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), or double-stranded DNA/RNA hybrids. Trigger polynucleotides may comprise naturally-occurring nucleotides, modified nucleotides, nucleotide analogues or any combination thereof. In some embodiments, a trigger polynucleotide may be incorporated within a larger polynucleotide. In some embodiments, a trigger polynucleotide may be processed into a small interfering RNA (siRNA). A trigger as disclosed herein includes, without limitation, a directional trigger, a directional chimeric trigger, and a strand-selective directional chimeric trigger.
As used herein, a directional trigger is an exogenous dsRNA molecule which can cause the silencing of at least one target gene, and has a preferential directionality when processed into small RNAs by a Dicer-like protein. One embodiment of a directional trigger has a 3' overhang and a 5' overhang on the same strand which, without being bound to any scientific theory or mechanism, favors the initiation of dicer processing from the 3' end and disfavors the initiation of dicer processing from the 5' end. In some embodiments, a directional trigger is a chimeric trigger, which comprises two or more target-specific sequences that, when the directional trigger is cleaved by a Dicer-like protein, yield predictable siRNAs, each of which has one target-specific sequence.
As used herein, a strand-selective directional chimeric trigger is a directional chimeric trigger capable of producing two or more sRNA duplexes, a majority of which sRNA duplexes preferentially have their antisense strands as guide strands.
A non-directional dsRNA trigger molecule ("non-directional trigger") is a dsRNA molecule which has no preferential directionality when processed into sRNAs by a dicer-like protein. Embodiments of a non-directional trigger include, but are not limited to, a dsRNA trigger molecule lacking a 3' initiator overhang, a 5' blocker overhang, or both.
As used herein, the terms "essentially identical" or "essentially complementary" means that the trigger (or at least one strand of a double-stranded polynucleotide or portion thereof, or a portion of a single strand polynucleotide) hybridizes under physiological conditions to the target gene, an RNA transcribed there from, or a fragment thereof, to effect regulation or suppression of the target gene. For example, in some embodiments, a trigger (or at least one strand of a double-stranded trigger) has 100 percent sequence identity or at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to a sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more contiguous nucleotides in the target gene or RNA transcribed from the target gene. In some embodiments, a trigger (or at least one strand of a double-stranded trigger) has 100 percent sequence complementarity or at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity when compared to a sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more contiguous nucleotides in the target gene or RNA transcribed from the target gene. In some embodiments, a trigger (or at least one strand of a double-stranded trigger) has 100 percent sequence identity with or complementarity to one allele or one family member of a given target gene (coding or non-coding sequence of a gene). In some embodiments, a trigger (or at least one strand of a double-stranded trigger) has at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity with or complementarity to multiple alleles or family members of a given target gene. In some embodiments, a trigger (or at least one strand of a double-stranded trigger) has 100 percent sequence identity with or complementarity to multiple alleles or family members of a given target gene.
As used herein, with respect to a nucleic acid sequence, nucleic acid molecule, or a gene, the term "natural," "naturally existing," or "native" means that the respective sequence or molecule is present in a wild-type cell which has not been genetically modified or manipulated by man. A small RNA molecule naturally targeting a gene means a small RNA molecule present in a wild-type cell, and targeting a gene naturally occurring in the wild-type cell.
As used herein, the terms "homology" and "identity" when used in relation to nucleic acids, describe the degree of similarity between two or more nucleotide sequences. The percentage of "sequence identity" between two sequences is determined by comparing two optimally aligned sequences over a comparison window, such that the portion of the sequence in the comparison window may comprise additions or deletions (gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. A sequence that is identical at every position in comparison to a reference sequence is said to be identical to the reference sequence and vice-versa. An alignment of two or more sequences may be performed using any suitable computer program. For example, a widely used and accepted computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994).
As used herein, the term "high GC content" refers to at least 50% of guanine or cytosine in the nucleotide composition of a given nucleotide sequence.
As used herein, the terms "exogenous polynucleotide" and "exogenous nucleic acid molecule" relative to an organism refer to a heterologous nucleic acid molecule which does not naturally occur within that organism. An exogenous nucleic acid molecule may be introduced into an organism in a stable or transient manner. An exogenous nucleic acid molecule may comprise a nucleic acid sequence which is identical or partially homologous to an endogenous nucleic acid sequence of the organism.
As used herein, the terms "improving," "improved," "increasing," and "increased" refer to at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450% or greater increase in a property or attribute caused by a specific treatment or design feature.
As used herein, "a reduction" of the level of an agent such as a protein or mRNA means that the level is reduced relative to a cell or organism lacking a dsRNA molecule capable of reducing the agent.
As used herein, the term "at least a partial reduction" of the level of an agent such as a protein or mRNA means that the level is reduced at least 25% relative to a cell or organism lacking a dsRNA molecule capable of reducing the agent.
As used herein, "a substantial reduction" of the level of an agent such as a protein or mRNA means that the level is reduced relative to a cell or organism lacking a dsRNA molecule capable of reducing the agent, where the reduction of the level of the agent is at least 75%.
As used herein, "an effective elimination" of an agent such as a protein or mRNA is relative to a cell or organism lacking a dsRNA molecule capable of reducing the agent, where the reduction of the level of the agent is greater than 95%. An agent, preferably a dsRNA molecule, is preferably capable of providing at least a partial reduction, more preferably a substantial reduction, or most preferably effective elimination of another agent such as a protein or mRNA, wherein the agent leaves the level of a second agent essentially unaffected, substantially unaffected, or partially unaffected.
As used herein, the terms "suppress," "repress," "downregulate," and "silence," when referring to the expression or activity of a nucleic acid molecule in a cell, are used equivalently herein and mean that the level of expression or activity of the nucleic acid molecule in an organism or a cell after applying a method of the present disclosure is lower than its expression or activity in the organism or cell before applying the method, or compared to a control organism or cell lacking a nucleic acid molecule as disclosed herein.
As used herein, a "suppression," "repression," or "downregulation" of the level or activity of an agent such as a protein, mRNA, or RNA means that the level or activity is reduced relative to a substantially identical plant, part of a plant, or plant cell grown under substantially identical conditions, lacking a nucleic acid molecule as disclosed herein. As used herein, "suppression," "repression," or "downregulation" of the level or activity of an agent, such as, for example, a preRNA, mRNA, rRNA, tRNA, snoRNA, snRNA expressed by the target gene, and/or of the protein product encoded by it, means that the amount is reduced by 10% or more, for example, 20% or more, preferably 30% or more, more preferably 50% or more, even more preferably 70% or more, most preferably 80% or more, for example, 90%, relative to a cell or organism lacking a nucleic acid molecule as disclosed herein.
The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and isolated plant cells, tissues and organs. The plant may be in any form including, but not limited to, suspension cultures, endosperm, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. It will be appreciated, that the plant or seed thereof may be transgenic plants.
As used herein, the phrase "plant cell culture" refers to any type of native (naturally occurring) plant cells, plant cell lines and genetically modified plant cells, which are not assembled to form a complete plant, such that at least one biological structure of a plant is not present. Optionally, the plant cell culture of this embodiment of the present disclosure may comprise a particular type of a plant cell or a plurality of different types of plant cells. It should be noted that optionally plant cultures featuring a particular type of plant cell may be originally derived from a plurality of different types of such plant cells. In certain embodiments according to the present disclosure, the plant cell is a non-sexually reproducing plant cell. In other aspects, a plant cell of the present disclosure is a non-photosynthetic plant cell.
The processing of a dsRNA molecule as disclosed herein can be monitored using any methods or systems known in the art. In one aspect, the processing of a dsRNA molecule as disclosed herein is monitored in wheat germ extract (e.g., Promega Catalog # 1.4380). In another aspect, the processing of a dsRNA molecule as disclosed herein is monitored in plant protoplasts. In a further aspect, the processing of a dsRNA molecule as disclosed herein is monitored in a plant or parts thereof selected from the group consisting of suspension cultures, embryos, meristematic regions, calli, leaves, roots, shoots, flowers, fruits, seeds, gametophytes, sporophytes, pollen, and microspores.
In one aspect, the instant disclosure provides a double-stranded RNA (dsRNA) molecule comprising a). a first strand comprising in 5' to 3' direction i) a first sequence that is identical to at least 18 consecutive nucleotides of a first target nucleotide sequence; and ii) a second sequence that is identical to at least 18 consecutive nucleotides of a second target nucleotide sequence and b). a second strand comprising in the 5' to 3' direction, a 5'-overhang, a nucleotide sequence that is complementary to the first strand, and a 2 nucleotide 3'-overhang, wherein the 5'overhang is at least 5 nucleotides in length.
In some embodiments, the 5'-overhang of the second strand has a high GC content. In some embodiments, the 5 '-overhang of the second strand is 5 nucleotides in length. In one embodiment, the 5 '-overhang has the sequence GCGCG. In one embodiment, the 2 nucleotide 3 '-overhang of the second strand has the sequence UA.
In some embodiments, the first strand further comprises the nucleotides GCCAC located 3' to the nucleotide sequence that is essentially identical to at least 18 consecutive nucleotides of the target nucleotide sequence. In some embodiments, the first strand further comprises a 5' G. In a further embodiment, the 5' G is not identical to the target nucleotide sequence.
In some embodiments, the 2 nucleotide 3 '-overhang in the second strand comprises at least one modification that improves stability of the dsRNA molecule. In some embodiments, the 2 nucleotide 3 '-overhang comprises at least one modification selected from the group consisting of methylation, phosphorothioate addition, locked nucleic acids (LNAs), and any combination thereof.
In some embodiments, the target nucleotide sequence is a coding region of a mRNA, a 5' untranslated region, a 3' untranslated region, an intron, a promoter, an enhancer, a terminator, an rRNA, a tRNA, a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a non-coding RNA involved in RNA interference, and any combination thereof.
In some embodiments, the first strand of the dsRNA molecule further comprising in the 5' to 3' direction: a). a first sequence that is identical to at least 18 consecutive nucleotides of a first target nucleotide sequence; and b). a second sequence that is identical to at least 18 consecutive nucleotides of a second target nucleotide sequence and a second strand comprising in the 5' to 3' direction, a 5' overhang, a nucleotide sequence that is complementary to the first strand, and a 2 nucleotide 3'-overhang, wherein the 5'overhang is at least 5 nucleotides in length. In some embodiments, the first target nucleotide sequence and second target nucleotide sequence are from different genes. In other embodiments, the first target nucleotide sequence and second target nucleotide sequence are from the same gene. In some embodiments, the first and second target nucleotide sequences are identical. In some embodiments, the first target nucleotide sequence and second target nucleotide sequence are non-contiguous. In some embodiments, the first strand comprises one or more As between the first and second sequence. In one embodiment, the second sequence comprises a 5' G. In a further embodiment, the second sequence comprises a 5' GUA. In another embodiment, the second sequence comprises a 5' GAA. In one embodiment, the second sequence comprises a 3' AA.
In some embodiments, the 3' end of the first strand has a high GC content. In some embodiments, the 3' end of the first strand is not identical to the target nucleotide sequence.
In one aspect, the dsRNA molecule is processed to produce 21, 22, 23, and/or 24 nucleotide siRNAs. In some embodiments, the first and the second sequences in the first strand are 21 nucleotides in length.
In some embodiments, the first strand of the dsRNA further comprises a third sequence that is essentially identical to at least 18 consecutive nucleotides of a third target nucleotide sequence. In some embodiments, the first strand of the dsRNA further comprises a fourth sequence that is essentially identical to at least 18 consecutive nucleotides of a fourth target nucleotide sequence. In some embodiments, the first strand of the dsRNA further comprises a fifth sequence that is essentially identical to at least 18 consecutive nucleotides of a fifth target nucleotide sequence. In some embodiments, the first strand of the dsRNA further comprises a sixth sequence that is essentially identical to at least 18 consecutive nucleotides of a sixth target nucleotide sequence. In some embodiments, the first strand of the dsRNA further comprises a seventh sequence that is essentially identical to at least 18 consecutive nucleotides of a seventh target nucleotide sequence. In some embodiments, the first strand of the dsRNA further comprises a eighth sequence that is essentially identical to at least 18 consecutive nucleotides of a eighth target nucleotide sequence.
In some embodiments, the first strand of the dsRNA comprises multiple target-specific sequences that are essentially identical to at least 18 consecutive nucleotides of multiple target nucleotide sequences. In some embodiments, at least two, at least three, at least four, at least five, or at least six of the multiple target nucleotide sequences are from different genes. In some embodiments, at least two, at least three, at least four, at least five, or at least six of the multiple target nucleotide sequences are from the same gene.
In another aspect, the instant disclosure provides a dsRNA molecule comprising: a). a first strand comprising in the 5' to 3' direction, i). a first nucleotide sequence that is identical to at least 18 consecutive nucleotides of a first target- nucleotide sequence; ii). a second nucleotide sequence comprising 2 or more As; and iii). a third nucleotide sequence that is identical to at least 18 consecutive nucleotides of a second targeted nucleotide sequence or at least 18 consecutive nucleotides of the first target nucleotide sequence; and b). a second strand comprising in the 5' to 3' direction, a 5 nucleotide 5'-overhang, a nucleotide sequence that is complementary to the first strand, and a 2 nucleotide 3'-overhang. In some embodiments, the 5 nucleotide 5'-overhang has a high GC content. In one embodiment, the 5 nucleotide 5'-overhang has the sequence GCGCG. In one embodiment, the 2 nucleotide 3'-overhang has the sequence UC. In one embodiment, the first strand further comprises the nucleotides GCCAC located 3' to the third nucleotide sequence. In some embodiments, the first and the second target nucleotide sequences are selected from a coding region of a mRNA, a 5' untranslated region, a 3' untranslated region, an intron, a promoter, an enhancer, a terminator, an rRNA, a tRNA, a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a non-coding RNA involved in RNA interference, and any combination thereof. In some embodiments, the first target nucleotide sequence and the second target nucleotide sequence are from different genes. In some embodiments, the first target nucleotide sequence and the second target nucleotide sequence are from the same gene. In some embodiments, the first and second target nucleotide sequences are identical. In some embodiments, the 2 nucleotide 3'-overhang in the second strand comprises at least one modification that improves stability of the dsRNA molecule. In some embodiments, the 2 nucleotide 3'-overhang comprises at least one modification selected from the group consisting of methylation, phosphorothioate addition, locked nucleic acids (LNAs), and any combination thereof. Modifications can be introduced to the most 5' nucleotide for both strands and to all nucleotides being part of the overhangs. In one embodiment, all five nucleotides representing the 5' end of the antisense strand (5'- GCGCG-3') can be modified to enhance stability.
In some embodiments, the dsRNA molecule in the instant disclosure comprises a concatemer of identical trigger sequences. In some embodiments, the dsRNA molecule comprises at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight of identical trigger sequences.
In one aspect, the dsRNA molecule is processed to produce 21, 22, 23, and/or 24 nucleotide siRNAs. In some embodiments, the first and the third sequences in the first strand are 21 nucleotides in length.
In a further aspect, the instant disclosure also provides a composition comprising a dsRNA molecule disclosed herein.
In one aspect, the instant disclosure provides a method of regulating expression of at least one target gene, comprising applying onto the surface of a plant or plant part a composition comprising a dsRNA molecule disclosed herein, wherein the dsRNA molecule comprises a) a first strand comprising in the 5' to 3' direction i) a first sequence that is identical to at least 18 consecutive nucleotides of a first target nucleotide sequence and ii) a second sequence that is identical to at least 18 consecutive nucleotides of a second target nucleotide sequence and b) a second strand comprising in the 5' to 3' direction, a 5' overhang, a nucleotide sequence that is complementary to the first strand, and a 2 nucleotide 3' overhang, wherein the 5' overhang is at least 5 nucleotides in length. In one aspect, the dsRNA molecule transfers from the surface of the plant or plant part into a cell of the plant or plant part.
In some embodiments, , the first strand of the dsRNA molecule further comprises a third nucleotide sequence that is essentially identical to at least 18 consecutive nucleotides of a third target gene. In a further embodiment, the first strand of the dsRNA molecule further comprises a fourth nucleotide sequence that is essentially identical to at least 18 consecutive nucleotides of a fourth target gene.
In some embodiments, the first strand of the dsRNA molecule comprises multiple target-specific sequences that are essentially identical to at least 18 consecutive nucleotides of multiple target nucleotide sequences. In some embodiments, at least two, at least three, at least four, at least five, or at least six of the multiple target nucleotide sequences are from different genes. In some embodiments, at least two, at least three, at least four, at least five, or at least six of the multiple target nucleotide sequences are from the same gene.
In some embodiments, the dsRNA molecule suppresses the expression of at least one, at least two, at least three, at least four, at least five, or at least six target genes.
In another aspect, the instant disclosure also provides a method of improving the efficiency of a dsRNA molecule in producing desired small RNAs in a plant or plant part, comprising providing to the plant or plant part a dsRNA molecule disclosed herein, wherein the production of the 21-24 nucleotide small RNAs is directionally biased towards the 3' end of the second strand of the dsRNA molecule.
In another aspect, the instant disclosure provides a plant, plant part, or seed comprising a dsRNA molecule disclosed herein, wherein the dsRNA molecule is exogenous to the plant, plant part, or seed. In one aspect, the dsRNA molecule suppresses the expression of at least one, at least two, at least three, or at least four target genes in the plant, plant part, or seed.
The instant disclosure provides a directional trigger comprising an exogenous dsRNA molecule having a preferential directionality when processed into sRNAs by a Dicer-like protein. In one aspect, a directional trigger of the instant disclosure comprises a 3' overhang. In another aspect, a directional trigger of the instant disclosure comprises a 5' overhang. In a further aspect, a directional trigger has a 3' overhang and a 5' overhang on the sense strand. In another aspect, a directional trigger has a 3' overhang and a 5' overhang on the antisense strand.
In one aspect, a directional trigger as disclosed herein has a pre-programmed processing pattern for generating sRNAs where sRNA processing starts from an end comprising a 3' overhang and continues in a phased manner with a about 21-nucleotide phase. In a further aspect, a directional trigger is capable of producing one or more sRNA duplexes that have strand-selectivity by preferentially having their antisense strands as guide strands. A guide strand of a sRNA duplex is the strand which is loaded into an RNA-induced silencing complex (RISC) and guides the recognition of a complementary mRNA molecule (e.g., target mRNA molecule) to trigger subsequent silencing.
In one aspect, a directional trigger as disclosed herein comprises two or more target-specific sequences that, when the directional trigger is cleaved by a Dicer-like protein, yield the same number of sRNAs, each of which has one target-specific sequence. In one aspect, two or more target-specific sequences are immediately adjacent to each other in a directional trigger. In one aspect, two or more target-specific sequences are not adjacent in a directional trigger. In one aspect, two or more target-specific sequences are not contiguous in a directional trigger. In one aspect, two or more target-specific sequences of a directional trigger are from two or more different genes. In another aspect, two or more target-specific sequences in a directional trigger are derived from a same gene but non-contiguous in that gene. In a further aspect, two or more target-specific sequences of a directional trigger have essentially identical sequences. In another aspect, a directional trigger further comprises one or more AU-rich linker sequences adjoining two or more target-specific sequences.
In one aspect, a dsRNA molecule or directional trigger as disclosed herein is not from a viral vector. In a further aspect, a dsRNA molecule or directional trigger as disclosed herein is not produced from a natural viral infection.
In one aspect, a dsRNA molecule or directional trigger as disclosed herein produces at least one, two, three, four, or five siRNAs (small interfering RNA). In one aspect, a dsRNA molecule or directional trigger as disclosed herein produces at least one, two, three, four, or five miRNAs (microRNAs). In one aspect, a dsRNA molecule or directional trigger as disclosed herein produces at least one, two, three, four, or five ta-siRNAs (trans activating siRNA). In one aspect, a dsRNA molecule or directional trigger as disclosed herein produces at least one, two, three, four, or five activating RNAs (RNAas). In one aspect, a dsRNA molecule or directional trigger as disclosed herein produces at least one, two, three, four, or five anti-sense siRNAs. In one aspect, a dsRNA molecule or directional trigger as disclosed herein produces at least one, two, three, four, or five hc-siRNAs (heterochromatic siRNAs). In one aspect, a dsRNA molecule or directional trigger as disclosed herein produces at least one, two, three, four, or five cis-acting siRNAs. In one aspect, a dsRNA molecule or directional trigger as disclosed herein produces at least one, two, three, four, or five ImiRNAs (long miRNAs). In one aspect, a dsRNA molecule or directional trigger as disclosed herein produces at least one, two, three, four, or five IsiRNAs (long siRNAs). In one aspect, a dsRNA molecule or directional trigger as disclosed herein produces at least one, two, three, four, or five easiRNAs (epigenetically activated siRNAs). In another aspect, a dsRNA molecule or directional trigger as disclosed herein produces at least one, two, three, four, or five sRNAs selected from the group consisting of siRNA, miRNA, ta-siRNA, RNAa, anti-sense siRNA, hc-siRNA, cis-acting siRNA, ImiRNA, IsiRNA, easiRNA and any combinations thereof.
A dsRNA molecule or directional trigger of the instant disclosure can be of variable length. In one aspect, each strand of a dsRNA molecule or directional trigger as disclosed herein has a length from about 20 to about 1000 nucleotides. In one aspect, each strand of a dsRNA molecule or directional trigger as disclosed herein has a length from about 25 to about 1000, from about 30 to about 1000, from about 35 to about 1000, from about 40 to about 1000, from about 45 to about 1000, from about 55 to about 1000, from about 60 to about 100, from about 65 to about 1000, from about 70 to about 1000, from about 75 to about 1000, from about 80 to about 1000, from about 85 to about 1000, from about 90 to about 1000, from about 95 to about 1000, from about 100 to about 1000, from about 150 to about 1000, from about 200 to about 1000, from about 250 to about 1000, from about 300 to about 1000, from about 350 to about 1000, from about 400 to about 1000, from about 500 to about 1000, from about 600 to about 1000, from about 700 to about 1000, from about 800 to about 1000, or from about 900 to about 1000 nucleotides.
In one embodiment, one strand of a dsRNA molecule or directional trigger has a length from about 20 to about 200, from about 25 to about 200, from about 30 to about 200, from about 35 to about 200, from about 40 to about 200, from about 45 to about 200, from about 50 to about 200, from about 55 to about 200, from about 60 to about 200, from about 65 to about 200, from about 70 to about 200, from about 75 to about 200, from about 80 to about 200, from about 85 to about 200, from about 90 to about 200, from about 95 to about 200, from about 100 to about 200, from about 105 to about 200, from about 110 to about 200, from about 120 to about 200, from about 130 to about 200, from about 140 to about 200, or from about 150 to about 200 nucleotides.
In another embodiment, one strand of a dsRNA molecule or directional trigger as disclosed herein has a length from about 20 to about 190, from about 25 to about 180, from about 30 to about 170, from about 35 to about 160, from about 40 to about 150, from about 45 to about 140, from about 50 to about 130, from about 55 to about 120, from about 60 to about 110, from about 65 to about 100, from about 70 to about 90, or from about 75 to about 80 nucleotides.
In one embodiment, one strand of a dsRNA molecule or directional trigger as disclosed herein has a length from about 40 to about 100, from about 45 to about 100, from about 50 to about 100, from about 55 to about 100, from about 60 to about 100, from about 65 to about 100, from about 70 to about 100, from about 75 to about 100, from about 80 to about 100, from about 85 to about 100, or from about 90 to about 100 nucleotides.
In another embodiment, one strand of a dsRNA molecule or directional trigger as disclosed herein has a length from about 40 to about 95, from about 40 to about 90, from about 40 to about 85, from about 40 to about 80, from about 40 to about 75, from about 40 to about 70, from about 40 to about 65, from about 40 to about 60, from about 40 to about 55, or from about 40 to about 50 nucleotides.
In a further embodiment, one strand of a dsRNA molecule or directional trigger as disclosed herein has a length from about 45 to about 95, from about 50 to about 90, from about 55 to about 85, from about 60 to about 80, or from about 65 to about 75.
In one embodiment, one strand of a dsRNA molecule or directional trigger as disclosed herein has a length from about 45 to about 75 nucleotides.
In another embodiment, one strand of a dsRNA molecule or directional trigger as disclosed herein has a length of about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides.
In one embodiment, a dsRNA molecule or directional trigger as disclosed herein comprises 2, 3, 4, 5, or 6 sRNA trigger sequences each encoding one sRNA duplex.
In one aspect, a dsRNA molecule or directional trigger as disclosed herein can comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatch regions. A mismatch region can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more sets of mismatched nucleotides. In another aspect, a dsRNA molecule or directional trigger as disclosed herein molecule can comprises one or more bulges of 1, 2, 3, 4 or more nucleotides.
In one embodiment, a dsRNA molecule or directional trigger of the instant disclosure has a single target gene. In another embodiment, a dsRNA molecule or directional trigger as disclosed herein has two or more distinct target genes. In one embodiment, two or more sRNA duplexes encoded by a dsRNA molecule or directional trigger of the instant disclosure target a same gene. A directional trigger as disclosed herein which encodes two or more distinct sRNAs targeting a same target gene can have a higher silencing efficiency compared to a non-directional trigger comprising similar target specific sequences with neither a 3' initiator overhang nor a 5' blocker overhang. Two silencing molecules' efficiencies can be compared by any available methods, for example, measuring minimum molecule concentrations needed to observe a set amount (e.g., 20%) of target gene expression reduction, or measuring percent reductions of target gene expression by applying a set amount (e.g., 250 pmol) of silencing molecules. One of ordinary skill in the art understands that both the set amount of target gene expression reduction and the set amount of silencing molecules can be varied when assessing the efficiency of different sets of molecules.
In another embodiment, two or more sRNA duplexes encoded by a dsRNA molecule or directional trigger of the instant disclosure do not target the same gene. In a further embodiment, each of two or more sRNA duplexes encoded by a dsRNA molecule or directional trigger of the instant disclosure targets a distinct gene. In another embodiment, a dsRNA molecule or directional trigger as disclosed herein targets two or more genes from the same gene family. In another embodiment, a dsRNA molecule or directional trigger as disclosed herein targets two or more paralogous genes. In another embodiment, a dsRNA molecule or directional trigger as disclosed herein targets two or more genes which are in a common metabolic pathway, and therefore increases the probability of disrupting the metabolic pathway. In another embodiment, a dsRNA molecule or directional trigger as disclosed herein targets two, three, or more separate herbicide-resistant genes.
In one embodiment, a dsRNA molecule or directional trigger as disclosed herein can comprise two or more target-specific sequences essentially identical or identical to a sequence (which can be coding sequence or non-coding sequence) selected from the group consisting of a plant endogenous gene sequence, a plant phytopathogen gene sequence, a plant viral gene sequence, a plant insect gene sequence, and combinations thereof. In one embodiment, a dsRNA molecule or directional trigger as disclosed herein can induce systemic regulation or suppression of an endogenous gene in a plant.
In one embodiment, a dsRNA molecule or directional trigger as disclosed herein has one or more target genes of interest which encode herbicide-tolerance proteins. Examples of a protein that provides tolerance to an herbicide include e.g., a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a glyphosate oxidoreductase (GOX), a glyphosate decarboxylase, a glyphosate-N-acetyl transferase (GAT), a dicamba monooxygenase, a phosphinothricin acetyltransferase, a 2,2- dichloropropionic acid dehalogenase, an acetohydroxyacid synthase, an acetolactate synthase, a haloarylnitrilase, an acetyl-coenzyme A carboxylase, a dihydropteroate synthase, a phytoene desaturase, a protoporphyrin IX oxygenase, a hydroxyphenylpyruvate dioxygenase, a para-aminobenzoate synthase, a glutamine synthase, a cellulose synthase, a beta-tubulin, and a serine hydroxymethyltransferase. Examples of nucleic acids encoding proteins conferring tolerance to herbicides include 5-enolpyruvylshikimate-3-phosphate synthases (EPSPS; see, e.g., U.S. Pat. Nos. 5,627,061 , 5,633,435 RE39,247 , 6,040,497 , and 5,094,945 , and PCT International Application Publications WO04074443 and WO04009761 ), glyphosate oxidoreductase (GOX; U.S. Pat. No. 5,463,175 ), glyphosate decarboxylase ( PCT International Application Publication WO05003362 , U.S. Pat. No. 7,405,347 , and U.S. Patent Application Publication 2004/0177399 ), glyphosate-N-acetyl transferase (GAT; U.S. Pat. No. 7,714,188 ) conferring tolerance to glyphosate; dicamba monooxygenase conferring tolerance to auxin-like herbicides such as dicamba ( U.S. Pat. No. 7,105,724 ); phosphinothricin acetyltransferase (pat or bar) conferring tolerance to phosphinothricin or glufosinate ( U.S. Pat. No. 5,646,024 ); 2,2-dichloropropionic acid dehalogenase conferring tolerance to 2,2-dichloropropionic acid (Dalapon) ( PCT International Application Publication WO9927116 ); acetohydroxyacid synthase or acetolactate synthase conferring tolerance to acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide ( U.S. Pat. No. 6,225,105 ); haloarylnitrilase (Bxn) for conferring tolerance to bromoxynil ( U.S. Pat. No. 4,810,648 ); modified acetyl-coenzyme A carboxylase for conferring tolerance to cyclohexanedione (sethoxydim) and aryloxyphenoxypropionate (haloxyfop) ( U.S. Pat. No. 6,414,222 ); dihydropteroate synthase (sul I) for conferring tolerance to sulfonamide herbicides ( U.S. Pat. No. 5,719,046 ); 32 kDa photosystem II polypeptide (psbA) for conferring tolerance to triazine herbicides (Hirschberg et al., 1983, Science, 222:1346-1349); anthranilate synthase for conferring tolerance to 5-methyltryptophan ( U.S. Pat. No. 4,581,847 ); dihydrodipicolinic acid Synthase (dap A) for conferring to tolerance to aminoethyl cysteine ( PCT International Application Publication WO8911789 ); phytoene desaturase (crtI) for conferring tolerance to pyridazinone herbicides such as norflurazon (Japan Patent JP06343473 ); hydroxyphenylpyruvate dioxygenase, a 4-hydroxyphenylacetic acid oxidase and a 4-hydroxyphenylacetic I-hydrolase ( U.S. Pat. No. 7,304,209 ) for conferring tolerance to cyclopropylisoxazole herbicides such as isoxaflutole ( U.S. Pat. No. 6,268,549 ); modified protoporphyrinogen oxidase I (protox) for conferring tolerance to protoporphyrinogen oxidase inhibitors ( U.S. Pat. No. 5,939,602 ); aryloxyalkanoate dioxygenase (AAD-I) for conferring tolerance to an herbicide containing an aryloxyalkanoate moiety ( PCT International Application Publication WO05107437 ); a serine hydroxymethyltransferase ( U.S. Patent Application Publication 2008/0155716 ), a glufosinate-tolerant glutamine synthase ( U.S. Patent Application Publication 2009/0018016 ). Examples of such herbicides include phenoxy auxins (such as 2,4-D and dichlorprop), pyridyloxy auxins (such as fluroxypyr and triclopyr), aryloxyphenoxypropionates (AOPP) acetyl-coenzyme A carboxylase (ACCase) inhibitors (such as haloxyfop, quizalofop, and diclofop), and 5-substituted phenoxyacetate protoporphyrinogen oxidase 1^x inhibitors (such as pyraflufen and flumiclorac)..
In another embodiment, a dsRNA molecule or directional trigger as disclosed herein has one or more target genes of interest which are essential genes. Essential genes are genes necessary for sustaining cellular life or to support reproduction of an organism. Exemplary essential genes include genes involved in DNA or RNA replication, gene transcription, RNA- mediated gene regulation, protein synthesis, energy production, and cell division. One example of a compendium of essential genes is described in Zhang et al. (2004) Nucleic Acids Res., 32:D271-D272 (listing 777 essential genes for Arabidopsis thaliana). Further examples of essential genes include translation initiation factor (TIF) and ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO). In further aspects, target genes of interest can be genes encoding transcription factors and genes encoding enzymes involved in the biosynthesis or catabolism of molecules in plants such as, but not limited to, amino acids, fatty acids and other lipids, sugars and other carbohydrates, biological polymers, and secondary metabolites including alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin.
In some embodiments, a target-specific sequence comprised in a nucleic acid molecule as disclosed herein has 100 % sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence complementarity when compared to a region of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more contiguous nucleotides in the target gene or RNA transcribed from the target gene. In some embodiments, a target-specific sequence comprised in a nucleic acid molecule as disclosed herein has 100 % sequence complementarity to one allele or one family member of a given target gene (coding or non-coding sequence of a gene). In some embodiments, a target-specific sequence comprised in a nucleic acid molecule as disclosed herein has at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence complementarity to multiple alleles or family members of a given target gene. In some embodiments, a target-specific sequence comprised in a nucleic acid molecule has 100% sequence complementarity to multiple alleles or family members of a given target gene.
In some embodiments, a target-specific sequence comprised in a nucleic acid molecule as disclosed herein has 100 % sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity when compared to a region of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more contiguous nucleotides in the target gene or RNA transcribed from the target gene. In some embodiments, a target-specific sequence comprised in a nucleic acid molecule as disclosed herein has 100 % sequence identity with one allele or one family member of a given target gene (coding or non-coding sequence of a gene). In some embodiments, a target-specific sequence comprised in a nucleic acid molecule as disclosed herein has at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with multiple alleles or family members of a given target gene. In some embodiments, a target-specific sequence comprised in a nucleic acid has 100 % sequence identity with multiple alleles or family members of a given target gene.
In one aspect, a dsRNA molecule or directional trigger of the instant disclosure can effect regulation of gene expression (e.g., suppression) for a time period of various length. In one aspect, a dsRNA molecule or directional trigger as disclosed herein is effective for a period during the life of a treated plant of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks. In another aspect, a dsRNA molecule or directional trigger as disclosed herein can be applied to a seed and subsequently regulate gene expression at any stage after the seed germinates. A seed treated with a dsRNA molecule or directional trigger as disclosed herein can be stored for a period of any length, e.g., 2, 4, or 6 weeks, or 2, 3, 4, 5, 6, 7, 8, 9, 10, I1, 12, 13, 14, 15, 16, 17, 18, 19, 20 months, or 2, 3, 4, 5, 6, 7 or more years, while retaining effects of the dsRNA molecule or directional trigger.
A dsRNA molecule or directional trigger of the instant disclosure can comprise a 3' overhang of variable length. In one aspect, a dsRNA molecule or directional trigger as disclosed herein comprises a 3' overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. In another aspect, a 3' overhang of a dsRNA molecule or directional trigger as disclosed herein comprise a non-target-specific sequence. In another aspect, a 3' overhang of a dsRNA molecule or directional trigger as disclosed herein comprise a synthetic sequence. In one aspect, a 3' overhang of a dsRNA molecule or directional trigger as disclosed herein comprises a sequence of 5'-Uracile-Adenine-3'. In another aspect, a 3' overhang of a dsRNA molecule or directional trigger as disclosed herein comprises a sequence of 5'-Uracile-Uracile-3'. Without being bound to any scientific theory or mechanism, siRNAs with 3' overhanging UU di-nucleotides were observed to be the most effective in triggering silencing. See Elbashir et al., Functional anatomy of siRNA for mediating efficient RNAi in Drosophila melanogaster embryo lysate, EMBO J, 20:6877-6888(2001); see also Strapps et al. The siRNA sequence and guide strand overhangs are determinants of in vivo duration of silencing, Nucleic Acids Research, 38(14):4799-97(2010). In another aspect, a 3' overhang of a dsRNA molecule or directional trigger as disclosed herein does not comprise a Guanine residue. Without being bound to any scientific theory or mechanism, RNase may cleave a siRNA at a single-stranded Guanine residue. In another aspect, the first non-overhung nucleotide immediately next to a 3' overhang of a dsRNA molecule or directional trigger as disclosed herein comprise a Cytosine. In a further aspect, a 3' overhang end of any nucleic acid molecule as disclosed herein can be substituted or combined with a frayed end.
In one aspect, a dsRNA molecule or directional trigger of the instant disclosure is diced by a Dicer-like protein preferentially at a distance of about 21 nucleotides from the terminus of a 3' overhang end creating a first sRNA duplex, wherein dicing of the dsRNA molecule or directional trigger continues in the same orientation at an interval length of about 21 nucleotides generating a second sRNA duplexes, and both the first and second sRNA duplexes are about 21 nucleotides long with a double-stranded region of about 19 nucleotides and a 2-nucleotide 3' overhang at both ends.
In one aspect, a dsRNA molecule or directional trigger of the instant disclosure is diced by a Dicer-liker protein preferentially at a distance of about 21 nucleotides from an end with a 3' overhang creating a first sRNA duplex, wherein dicing of the dsRNA molecule or directional trigger continues in the same orientation at an interval length of about 21 nucleotides generating one or more successive sRNA duplexes, and wherein the first and one or more successive sRNA duplexes are about 21 nucleotides long with a double-stranded region of about 19 nucleotides and a 2-nucleotide 3' overhang at both ends. In one aspect, a dsRNA molecule or directional trigger of the instant disclosure can be diced into at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more successive sRNA duplexes of about 21 nucleotides with a double-stranded region of about 19 nucleotides and a 2-nucleotide 3' overhang at both ends.
A dsRNA molecule or directional trigger of the instant disclosure can comprise at one end a 5' overhang of variable length which, without being to any theory or mechanism, substantially disfavors or essentially precludes a Dicer-liker protein to start dicing from that end. In one aspect, a dsRNA molecule or directional trigger as disclosed herein comprise a 5' overhang of 5, 6, 7, 8, 9, 10 or more nucleotides. In another aspect, a 5' overhang of a dsRNA molecule or directional trigger as disclosed herein comprise a non-target-specific sequence. In another aspect, a 5' overhang of a dsRNA molecule or directional trigger as disclosed herein comprise a synthetic sequence. In a further aspect, a 5' overhang of a dsRNA molecule or directional trigger as disclosed herein comprises a sequence of 5 '-Guanine-Guanine-Guanine-3'. In a further aspect, a 5' overhang end of any nucleic acid molecule as disclosed herein can be substituted or combined with a frayed end.
In one aspect, a dsRNA molecule or directional trigger as disclosed herein comprises, immediately next to a 5' overhang, a double-stranded region having a non-target-specific sequence of variable length. In one aspect, a 5' overhang-adjacent non-target-specific double-stranded region comprises a length selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides.
In one aspect, a dsRNA molecule or directional trigger as disclosed herein comprises one or more modified nucleotides. In another aspect, a 5' overhang of a dsRNA molecule or directional trigger as disclosed herein comprises 5, 6, 7, 8, 9, 10 or more deoxyribonucleotides. In another aspect, a 5' overhang of a dsRNA molecule or directional trigger as disclosed herein is solely composed of deoxyribonucleotides.
A dsRNA molecule or directional trigger of the instant disclosure can also comprise a linker sequence of variable length adjoining two adjacent sRNA trigger sequences. In one aspect, a linker sequence as disclosed herein is about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides long.
In one aspect, a linker sequence as disclosed herein comprises a non-target-specific sequence. In another aspect, a linker sequence as disclosed herein comprises a synthetic sequence. In another aspect, a linker sequence as disclosed herein is rich in Adenine or Uracil. In one aspect, at least 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the sequence of a linker used here comprises Adenine (A) or Uracil (U). In another aspect, a linker sequence as disclosed herein comprises a sequence of 5'-AAAAG-3' (SEQ ID NO:80).
A dsRNA molecule or directional trigger of the instant disclosure can further be a strand-selective trigger or chimera encoding one or more sRNA duplexes which preferentially have their antisense strands as guide strands. In one aspect, at least a most abundant sRNA duplex produced from a dsRNA molecule or directional trigger of the instant disclosure preferentially have their antisense strands as guide strands. In another aspect, both a most abundant and a second most abundant sRNA duplex produced from a dsRNA molecule or directional trigger of the instant disclosure preferentially have their antisense strands as guide strands. In a further aspect, both a most abundant, a second most abundant, and a third most abundant sRNA duplex produced from a dsRNA molecule or directional trigger of the instant disclosure preferentially have their antisense strands as guide strands. In a further aspect, both a most abundant, a second most abundant, a third most abundant, a fourth most abundant sRNA duplex produced from a dsRNA molecule or directional trigger of the instant disclosure preferentially have their antisense strands as guide strands. A guide strand of a sRNA duplex is the strand which is loaded into a RNA-induced silencing complex (RISC) to guide the recognition of a complementary mRNA molecule and to trigger subsequent silencing.
In one aspect, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sRNA duplexes encoded by a strand-selective trigger as disclosed herein have their antisense strands as guide strands. In another aspect, less than 10%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of sRNA duplexes encoded by a strand-selective trigger as disclosed herein have their sense strands as guide strands.
In one aspect, a sRNA duplex encoded by a dsRNA molecule or directional trigger as disclosed herein comprises a Uracil or Cytosine at the 5' end of its antisense strand. In another aspect, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of sRNA duplexes encoded by a dsRNA molecule or directional trigger as disclosed herein comprise a Uracil or Cytosine at the 5' end of their antisense strands.
In another aspect, a sRNA duplex encoded by a dsRNA molecule or directional trigger of the instant disclosure comprises a Guanine at the 5' end of its sense strand. In a further aspect, a sRNA duplex encoded by a dsRNA molecule or directional trigger of the instant disclosure comprises a Uracil or Cytosine at the 5' end of its antisense strand, and a Guanine at the 5' end of its sense strand. In another aspect, a dsRNA molecule or directional trigger as disclosed herein comprises a Uracil or Cytosine at position 21 on a same strand bearing a 2-nucleotide 3' overhang, where position 21 is the 21 st nucleotide relative to the terminus of the 2-nucleotide 3' overhang.
A dsRNA molecule or directional trigger of the instant disclosure can be processed into a more homogenous population of sRNAs compared to a non-directional trigger. In one aspect, a dsRNA molecule or directional trigger as disclosed herein produce sRNAs predominately originating from a specific region or strand of the dsRNA molecule or directional trigger. In one aspect, sRNA produced by a dsRNA molecule or directional trigger as disclosed herein originate primarily from the 3' end of the antisense strand of the dsRNA molecule or directional trigger. In another aspect, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of total sRNAs produced by a dsRNA molecule or directional trigger as disclosed herein originate primarily from the 3' end of the antisense strand of the dsRNA molecule or directional trigger. In a further aspect, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of total sRNAs of 21-24nt produced by a dsRNA molecule or directional trigger as disclosed herein originate primarily from the 3' end of the antisense strand of the dsRNA molecule or directional trigger.
A dsRNA molecule or directional trigger as disclosed herein can have an increased silencing efficiency compared to a non-directional trigger comprising similar or essentially identical target specific sequences. In one aspect, a dsRNA molecule or directional trigger as disclosed herein can achieve a percent reduction of target gene expression that is similar to, substantially same or same as that achieved by a non-directional trigger when the dsRNA molecule or directional trigger is applied at a concentration of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of that of the non-directional trigger.
A dsRNA molecule or directional trigger of the instant disclosure can be applied to a plant, plant part or seed at any concentration needed to effect a desirable degree of silencing of its target(s). In one aspect, a dsRNA molecule or directional trigger as disclosed herein can result in at least a partial reduction, a substantial reduction, an effective elimination or a suppression of target gene expression when applied to a plant or seed at a concentration selected from the group consisting of less than about 100, 75, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1, 0.05, and 0.01 µM.
In one aspect, a dsRNA molecule or directional trigger as disclosed herein can result in at least a partial reduction, a substantial reduction, an effective elimination or a suppression of target gene expression when applied to a plant or seed at a concentration selected from the group consisting of between 0.1 and 10, between 0.2 and 10, between 0.3 and 10, between 0.4 and 10, between 0.5 and 10, between 0.6 and 10, between 0.7 and 10, between 0.8 and 10, between 0.9 and 10, between 1 and 10, between 2 and 10, between 3 and 10, between 4 and 10, between 5 and 10, between 6 and 10, between 7 and 10, between 8 and 10, and between 9 and 10 µM.
In another aspect, a dsRNA molecule or directional trigger as disclosed herein can result in at least a partial reduction, a substantial reduction, an effective elimination or a suppression of target gene expression when applied to a plant or seed at a concentration selected from the group consisting of between 0.1 and 10, between 0.2 and 9, between 0.3 and 8, between 0.4 and 7, between 0.5 and 6, between 0.6 and 5, between 0.7 and 4, between 0.8 and 3, and between 0.9 and 2 µM.
In a further aspect, a dsRNA molecule or directional trigger as disclosed herein can result in at least a partial reduction, a substantial reduction, an effective elimination or a suppression of target gene expression when applied to a plant or seed at a concentration selected from the group consisting of between 0.1 and 10, between 0.1 and 9, between 0.1 and 8, between 0.1 and 7, between 0.1 and 6, between 0.1 and 5, between 0.1 and 4, between 0.1 and 3, between 0.1 and 2, between 0.1 and 1, between 0.1 and 0.9, between 0.1 and 0.8, between 0.1 and 0.7, between 0.1 and 0.6, between 0.1 and 0.5, between 0.1 and 0.4, between 0.1 and 0.3, and between 0.1 and 0.2 µM.
In one aspect, the instant disclosure provides a dsRNA molecule, wherein the processing of a population of the dsRNA molecules into one or more 21-24mer sRNAs preferentially starts from an end having a 3' overhang, and wherein at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of detectable 21-24mer sRNAs processed therefrom comprise a sequence identical to a sequence of a first double-stranded portion of at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides immediately adjacent to the 3' overhang. In one aspect, the dsRNA molecule is a directional trigger. In one aspect, the 3' overhang has a length of 2, or 3 nucleotides. In another aspect, the 3' overhang has a length of more than 3 nucleotides. In another aspect, the dsRNA molecule further comprises a 5' overhang. In another aspect, the 5' overhang is on the same strand having the 3' overhang. In a further aspect, the 5' overhang is 5 nucleotides long. In another aspect, the 5' overhang comprises deoxyribonucleotides. In another aspect, the 5' overhang comprises one, two, or three Guanine at the terminus. In a further aspect, the dsRNA molecule has a length between about 45 and about 75 nucleotides. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are not found in a single naturally occurring dsRNA molecule. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are not contiguous in a single naturally occurring molecule. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are adjoined by one or more synthetic linker sequences. In one aspect, the linker sequences are about 5 nucleotides long. In another aspect, the linker sequences are Adenine- or Uracil-rich sequences. In one aspect, the two or more sRNA duplexes target a same gene. In another aspect, the two or more sRNA duplexes do not target a same gene. In a further aspect, each of the two or more sRNA duplexes targets a distinct gene. In one aspect, the guide strand of each of the two or more sRNA duplexes is from the same strand of the dsRNA molecule. In another aspect, the guide strand of each of the two or more populations of sRNAs is from the antisense strand of the dsRNA molecule. In a further aspect, the guide strand of each of the two or more sRNA duplexes is from opposite strands of the dsRNA molecule. In a further aspect, each of the two or more sRNA trigger sequences has a length between about 20 and about 30 nucleotides. In one aspect, the dsRNA molecule comprises a Uracil at position 21 on the same strand bearing the 3' overhang, the position 21 is the 21 st nucleotides relative to terminus of the 3' overhang. In another aspect, wherein the dsRNA molecule further comprises a Uracil at position 20 on the same strand bearing the 3' overhang, the position 20 is the 20th nucleotides relative to terminus of the 3' overhang. In a further aspect, the 5' terminal nucleotide of the 3' overhang end of the dsRNA molecule is Guanine.
In one aspect, the instant disclosure provides a dsRNA molecule, wherein the first cleavage of the dsRNA molecule by a Dicer-like protein is at a position of about 21 to 24 nucleotides from the 3' terminus of a 3' overhang, and wherein at least 50%, 60%, 65%, 70%,, 75%, 80%, 85%, 90%, or 95% of detectable 21-24mer sRNAs processed from the population of the dsRNA molecules comprise a sequence identical to a sequence of a first double-stranded portion of at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides immediately adjacent to the 3' overhang. In one aspect, the dsRNA molecule is a directional trigger. In one aspect, the 3' overhang has a length of 2, or 3 nucleotides. In another aspect, the 3' overhang has a length of more than 3 nucleotides. In another aspect, the dsRNA molecule further comprises a 5' overhang. In another aspect, the 5' overhang is on the same strand having the 3' overhang. In a further aspect, the 5' overhang is 5 nucleotides long. In another aspect, the 5' overhang comprises deoxyribonucleotides. In another aspect, the 5' overhang comprises one, two, or three Guanine at the terminus. In a further aspect, the dsRNA molecule has a length between about 45 and about 75 nucleotides. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are not found in a single naturally occurring dsRNA molecule. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are not contiguous in a single naturally occurring molecule. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are adjoined by one or more synthetic linker sequences. In one aspect, the linker sequences are about 5 nucleotides long. In another aspect, the linker sequences are Adenine- or Uracil-rich sequences. In one aspect, the two or more sRNA duplexes target a same gene. In another aspect, the two or more sRNA duplexes do not target a same gene. In a further aspect, each of the two or more sRNA duplexes targets a distinct gene. In one aspect, the guide strand of each of the two or more sRNA duplexes is from the same strand of the dsRNA molecule. In another aspect, the guide strand of each of the two or more populations of sRNAs is from the antisense strand of the dsRNA molecule. In a further aspect, the guide strand of each of the two or more sRNA duplexes is from opposite strands of the dsRNA molecule. In a further aspect, each of the two or more sRNA trigger sequences has a length between about 20 and about 30 nucleotides. In one aspect, the dsRNA molecule comprises a Uracil at position 21 on the same strand bearing the 3' overhang, the position 21 is the 21 st nucleotide relative to terminus of the 3' overhang. In another aspect, wherein the dsRNA molecule further comprises a Uracil at position 20 on the same strand bearing the 3' overhang, the position 20 is the 20th nucleotide relative to terminus of the 3' overhang. In a further aspect, the 5' terminal nucleotide of the 3' overhang end of the dsRNA molecule is Guanine.
In one aspect, the instant disclosure provides a dsRNA molecule comprising a 5' overhang, wherein the first cleavage of the dsRNA molecule by a Dicer-like protein is at a position of about 21 to 24 nucleotides from the 3' terminus of the dsRNA molecule, and wherein at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of detectable 21-24mer sRNAs processed from the population of the dsRNA molecules comprise a sequence identical to a sequence of a first double-stranded portion of at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides. In one aspect, the dsRNA molecule is a directional trigger. In one aspect, the 3 'terminus comprises a 3' overhang having a length of 2, or 3 nucleotides. In another aspect, the 3' overhang has a length of more than 3 nucleotides. In another aspect, the 5' overhang is on the same strand having the 3' overhang. In a further aspect, the 5' overhang is 5 nucleotides long. In another aspect, the 5' overhang comprises deoxyribonucleotides. In another aspect, the 5' overhang comprises one, two, or three Guanine at the terminus. In a further aspect, the dsRNA molecule has a length between about 45 and about 75 nucleotides. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are not found in a single naturally occurring dsRNA molecule. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are not contiguous in a single naturally occurring molecule. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are adjoined by one or more synthetic linker sequences. In one aspect, the linker sequences are about 5 nucleotides long. In another aspect, the linker sequences are Adenine- or Uracil-rich sequences. In one aspect, the two or more sRNA duplexes target a same gene. In another aspect, the two or more sRNA duplexes do not target a same gene. In a further aspect, each of the two or more sRNA duplexes targets a distinct gene. In one aspect, the guide strand of each of the two or more sRNA duplexes is from the same strand of the dsRNA molecule. In another aspect, the guide strand of each of the two or more populations of sRNAs is from the antisense strand of the dsRNA molecule. In a further aspect, the guide strand of each of the two or more sRNA duplexes is from opposite strands of the dsRNA molecule. In a further aspect, each of the two or more sRNA trigger sequences has a length between about 20 and about 30 nucleotides. In one aspect, the dsRNA molecule comprises a Uracil at position 21 on the same strand bearing the 5' overhang, the position 21 is the 21 st nucleotide relative to 3' terminus of the dsRNA molecule. In another aspect, wherein the dsRNA molecule further comprises a Uracil at position 20 on the same strand bearing the 5' overhang, the position 20 is the 20th nucleotide relative to terminus of the 3' terminus. In a further aspect, the 5' terminal nucleotide of the 3' overhang end of the dsRNA molecule is Guanine.
In one aspect, the instant disclosure provides a dsRNA molecule, wherein the processing of a population of the dsRNA molecule into one or more 21-24mer sRNAs preferentially starts from one end of the dsRNA molecule, and wherein the most abundant detectable 21-24mer sRNAs processed from the population of the dsRNA molecules comprise a sequence identical to a sequence of a first double-stranded portion of at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides immediately adjacent to the 3' overhang. In another aspect, the second most abundant detectable 21-24mer sRNAs processed from a population of dsRNA molecules as disclosed herein comprise a sequence identical to a sequence immediately adjacent to the first double-stranded portion. In one aspect, the dsRNA molecule is a directional trigger. In one aspect, the 3' overhang has a length of 2, or 3 nucleotides. In another aspect, the 3' overhang has a length of more than 3 nucleotides. In another aspect, the dsRNA molecule further comprises a 5' overhang. In another aspect, the 5' overhang is on the same strand having the 3' overhang. In a further aspect, the 5' overhang is 5 nucleotides long. In another aspect, the 5' overhang comprises deoxyribonucleotides. In another aspect, the 5' overhang comprises one, two, or three Guanine at the terminus. In a further aspect, the dsRNA molecule has a length between about 45 and about 75 nucleotides. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are not found in a single naturally occurring dsRNA molecule. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are not contiguous in a single naturally occurring molecule. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are adjoined by one or more synthetic linker sequences. In one aspect, the linker sequences are about 5 nucleotides long. In another aspect, the linker sequences are Adenine- or Uracil-rich sequences. In one aspect, the two or more sRNA duplexes target a same gene. In another aspect, the two or more sRNA duplexes do not target a same gene. In a further aspect, each of the two or more sRNA duplexes targets a distinct gene. In one aspect, the guide strand of each of the two or more sRNA duplexes is from the same strand of the dsRNA molecule. In another aspect, the guide strand of each of the two or more populations of sRNAs is from the antisense strand of the dsRNA molecule. In a further aspect, the guide strand of each of the two or more sRNA duplexes is from opposite strands of the dsRNA molecule. In a further aspect, each of the two or more sRNA trigger sequences has a length between about 20 and about 30 nucleotides. In one aspect, the dsRNA molecule comprises a Uracil at position 21 on the same strand bearing the 3' overhang, the position 21 is the 21 st nucleotide relative to terminus of the 3' overhang. In another aspect, wherein the dsRNA molecule further comprises a Uracil at position 20 on the same strand bearing the 3' overhang, the position 20 is the 20th nucleotide relative to terminus of the 3' overhang. In a further aspect, the 5' terminal nucleotide of the 3' overhang end of the dsRNA molecule is Guanine.
In one aspect, the instant disclosure provides a dsRNA molecule comprising a 5' overhang at a first end of the dsRNA molecule, wherein the processing of the dsRNA molecule into one or more sRNAs preferentially starts from a second end of the dsRNA molecule, and wherein the first and second ends are opposite ends of the dsRNA molecule. In one aspect, the dsRNA molecule is a directional trigger. In one aspect, the second end comprises a 3' overhang. In one aspect, the 3' overhang has a length of 2, or 3 nucleotides. In another aspect, the 3' overhang has a length of more than 3 nucleotides. In another aspect, the dsRNA molecule further comprises a 5' overhang. In another aspect, the 5' overhang is on the same strand having the 3' overhang. In a further aspect, the 5' overhang is 5 nucleotides long. In another aspect, the 5' overhang comprises deoxyribonucleotides. In another aspect, the 5' overhang comprises one, two, or three Guanine at the terminus. In a further aspect, the dsRNA molecule has a length between about 45 and about 75 nucleotides. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are not found in a single naturally occurring dsRNA molecule. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are not contiguous in a single naturally occurring molecule. In another aspect, the dsRNA molecule further comprises two or more sRNA trigger sequences that encode the same number of sRNA duplexes, wherein the two or more sRNA trigger sequences are adjoined by one or more synthetic linker sequences. In one aspect, the linker sequences are about 5 nucleotides long. In another aspect, the linker sequences are Adenine- or Uracil-rich sequences. In one aspect, the two or more sRNA duplexes target a same gene. In another aspect, the two or more sRNA duplexes do not target a same gene. In a further aspect, each of the two or more sRNA duplexes targets a distinct gene. In one aspect, the guide strand of each of the two or more sRNA duplexes is from the same strand of the dsRNA molecule. In another aspect, the guide strand of each of the two or more populations of sRNAs is from the antisense strand of the dsRNA molecule. In a further aspect, the guide strand of each of the two or more sRNA duplexes is from opposite strands of the dsRNA molecule. In a further aspect, each of the two or more sRNA trigger sequences has a length between about 20 and about 30 nucleotides. In one aspect, the dsRNA molecule comprises a Uracil at position 21 on the same strand bearing the 3' overhang, the position 21 is the 21 st nucleotide relative to terminus of the 3' overhang. In another aspect, wherein the dsRNA molecule further comprises a Uracil at position 20 on the same strand bearing the 3' overhang, the position 20 is the 20th nucleotide relative to terminus of the 3' overhang. In a further aspect, the 5' terminal nucleotide of the 3' overhang end of the dsRNA molecule is Guanine.
In one aspect, the instant disclosure provides a dsRNA molecule comprising two or more sRNA trigger sequences that encode the same number of sRNAs, and one or more Adenine- or Uracil-rich linker sequences adjoining the two or more sRNA trigger sequences, wherein the two or more sRNA trigger sequences are not found in a single naturally occurring molecule. In another aspect, the dsRNA molecule has a length between about 45 and about 75 nucleotides. In one aspect, the linker sequences are about 5 nucleotides long. In one aspect, the linker sequences are synthetic sequences. In one aspect, the two or more sRNA duplexes target a same gene. In another aspect, the two or more sRNA duplexes do not target a same gene. In a further aspect, each of the two or more sRNA duplexes targets a distinct gene. In one aspect, the guide strand of each of the two or more sRNA duplexes is from the same strand of the dsRNA molecule. In another aspect, the guide strand of each of the two or more populations of sRNAs is from the antisense strand of the dsRNA molecule. In a further aspect, the guide strand of each of the two or more sRNA duplexes is from opposite strands of the dsRNA molecule. In a further aspect, each of the two or more sRNA trigger sequences has a length between about 20 and about 30 nucleotides.
In one aspect, the instant disclosure provides a dsRNA molecule comprising two or more sRNA trigger sequences that encode the same number of sRNAs, and one or more Adenine- or Uracil-rich linker sequences adjoining the two or more sRNA trigger sequences, wherein the two or more sRNA trigger sequences are not contiguous in a single naturally occurring molecule. In another aspect, the dsRNA molecule has a length between about 45 and about 75 nucleotides. In one aspect, the linker sequences are synthetic sequences. In one aspect, the linker sequences are about 5 nucleotides long. In another aspect, the linker sequences are Adenine- or Uracil-rich sequences. In one aspect, the two or more sRNA duplexes target a same gene. In another aspect, the two or more sRNA duplexes do not target a same gene. In a further aspect, each of the two or more sRNA duplexes targets a distinct gene. In one aspect, the guide strand of each of the two or more sRNA duplexes is from the same strand of the dsRNA molecule. In another aspect, the guide strand of each of the two or more populations of sRNAs is from the antisense strand of the dsRNA molecule. In a further aspect, the guide strand of each of the two or more sRNA duplexes is from opposite strands of the dsRNA molecule. In a further aspect, each of the two or more sRNA trigger sequences has a length between about 20 and about 30 nucleotides.
In one aspect, the instant disclosure provides a dsRNA molecule comprising a 3' overhang and a 5' overhang, and further comprising two or more sRNA trigger sequences that encode the same number of sRNAs, wherein the two or more sRNA trigger sequences are not found in a single naturally occurring molecule. In another aspect, the dsRNA molecule has a length between about 45 and about 75 nucleotides. In one aspect, the dsRNA molecule is a directional trigger. In another aspect, the dsRNA molecule is a directional chimeric trigger. In a further aspect, the dsRNA molecule is a strand-selective chimeric trigger. In one aspect, the 3' overhang has a length of 2, or 3 nucleotides. In another aspect, the 3' overhang has a length of more than 3 nucleotides. In another aspect, the 5' overhang is on the same strand having the 3' overhang. In a further aspect, the 5' overhang is 5 nucleotides long. In another aspect, the 5' overhang comprises deoxyribonucleotides. In another aspect, the 5' overhang comprises one, two, or three Guanine at the terminus. In another aspect, the dsRNA molecule further comprises one or more synthetic linker sequences adjoining the two or more sRNA trigger sequences. In one aspect, the synthetic linker sequences are about 5 nucleotides long. In another aspect, the synthetic linker sequences are Adenine- or Uracil-rich sequences. In one aspect, the two or more sRNA duplexes target a same gene. In another aspect, the two or more sRNA duplexes do not target a same gene. In a further aspect, each of the two or more sRNA duplexes targets a distinct gene. In one aspect, the guide strand of each of the two or more sRNA duplexes is from the same strand of the dsRNA molecule. In another aspect, the guide strand of each of the two or more populations of sRNAs is from the antisense strand of the dsRNA molecule. In a further aspect, the guide strand of each of the two or more sRNA duplexes is from opposite strands of the dsRNA molecule. In a further aspect, each of the two or more sRNA trigger sequences has a length between about 20 and about 30 nucleotides. In one aspect, the dsRNA molecule comprises a Uracil at position 21 on the same strand bearing the 3' overhang, the position 21 is the 21 st nucleotide relative to terminus of the 3' overhang. In another aspect, wherein the dsRNA molecule further comprises a Uracil at position 20 on the same strand bearing the 3' overhang, the position 20 is the 20th nucleotide relative to terminus of the 3' overhang. In a further aspect, the 5' terminal nucleotide of the 3' overhang end of the dsRNA molecule is Guanine.
In one aspect, the instant disclosure provides a dsRNA molecule comprising a 3' overhang and a 5' overhang, and further comprising two or more sRNA trigger sequences that encode the same number of sRNAs, wherein the two or more sRNA trigger sequences are not contiguous in a single naturally occurring molecule. In another aspect, the dsRNA molecule has a length between about 45 and about 75 nucleotides. In one aspect, the dsRNA molecule is a directional trigger. In another aspect, the dsRNA molecule is a directional chimeric trigger. In a further aspect, the dsRNA molecule is a strand-selective chimeric trigger. In one aspect, the 3' overhang has a length of 2, or 3 nucleotides. In another aspect, the 3' overhang has a length of more than 3 nucleotides. In another aspect, the 5' overhang is on the same strand having the 3' overhang. In a further aspect, the 5' overhang is 5 nucleotides long. In another aspect, the 5' overhang comprises deoxyribonucleotides. In another aspect, the 5' overhang comprises one, two, or three Guanine at the terminus. In another aspect, the dsRNA molecule further comprises one or more synthetic linker sequences adjoining the two or more sRNA trigger sequences. In one aspect, the synthetic linker sequences are about 5 nucleotides long. In another aspect, the synthetic linker sequences are Adenine- or Uracil-rich sequences. In one aspect, the two or more sRNA duplexes target a same gene. In another aspect, the two or more sRNA duplexes do not target a same gene. In a further aspect, each of the two or more sRNA duplexes targets a distinct gene. In one aspect, the guide strand of each of the two or more sRNA duplexes is from the same strand of the dsRNA molecule. In another aspect, the guide strand of each of the two or more populations of sRNAs is from the antisense strand of the dsRNA molecule. In a further aspect, the guide strand of each of the two or more sRNA duplexes is from opposite strands of the dsRNA molecule. In a further aspect, each of the two or more sRNA trigger sequences has a length between about 20 and about 30 nucleotides. In one aspect, the dsRNA molecule comprises a Uracil at position 21 on the same strand bearing the 3' overhang, the position 21 is the 21 st nucleotide relative to terminus of the 3' overhang. In another aspect, wherein the dsRNA molecule further comprises a Uracil at position 20 on the same strand bearing the 3' overhang, the position 20 is the 20th nucleotide relative to terminus of the 3' overhang. In a further aspect, the 5' terminal nucleotide of the 3' overhang end of the dsRNA molecule is Guanine.
In one aspect, a dsRNA molecule or directional trigger of the instant disclosure comprises one, two, three, four, five or more features selected from the group consisting of (a) two or more sRNA trigger sequences each of which encodes a sRNA, where the two or more sRNA trigger sequences are not found in a single naturally occurring molecule or not contiguous in a single naturally occurring molecule, (b) having a length between about 45 and about 75 nucleotides, (c) comprising one or more Adenine- or Uracil-rich linker sequences adjoining the two or more sRNA trigger sequences, (d) comprising a 3' overhang in the antisense strand, (e) comprising a Uracil at positions 20 and 21 in the antisense strand relative to terminus of the 3' overhang, (f) a 5' overhang of 5 nucleotides, and (g) combinations thereof.
In one aspect, a dsRNA molecule or directional trigger of the instant disclosure comprises a first end portion comprising a 3' overhang, a second end portion comprising a 5' overhang, and two or more target-specific sequences that are adjoined by one or more linker sequences. In another aspect, the dsRNA molecule has a length between about 45 and about 75 nucleotides. In one aspect, the 3' overhang has a length of 2, or 3 nucleotides. In another aspect, the 3' overhang has a length of more than 3 nucleotides. In another aspect, the 5' overhang is on the same strand having the 3' overhang. In a further aspect, the 5' overhang is 5 nucleotides long. In another aspect, the 5' overhang comprises deoxyribonucleotides. In another aspect, the 5' overhang comprises one, two, or three Guanine at the terminus. In another aspect, the one or more linker sequences are synthetic sequences. In one aspect, the synthetic linker sequences are about 5 nucleotides long. In another aspect, the synthetic linker sequences are Adenine- or Uracil-rich sequences. In one aspect, the two or more target-specific sequences can be found in a naturally occurring molecule but not contiguous in that molecule. In another aspect, the two or more target-specific sequences are from different genes. In another aspect, the two or more target-specific sequences are from two different naturally occurring molecules. In one aspect, at least one of the two or more target-specific sequences has a length between about 20 and about 30 nucleotides. In a further aspect, each of the two or more target-specific sequences has a length between about 20 and about 30 nucleotides. In one aspect, the dsRNA molecule comprises a Uracil at position 21 on the same strand bearing the 3' overhang, the position 21 is the 21st nucleotide relative to terminus of the 3' overhang. In another aspect, wherein the dsRNA molecule further comprises a Uracil at position 20 on the same strand bearing the 3' overhang, the position 20 is the 20th nucleotide relative to terminus of the 3' overhang. In a further aspect, the 5' terminal nucleotide of the 3' overhang end of the dsRNA molecule is Guanine.
In one aspect, sRNAs produced by a dsRNA molecule or directional trigger as disclosed herein can regulate the expression of a target gene via any RNA silencing mechanism. Exemplary mechanisms include RNA cleavage, translation or transcription attenuation, DNA or chromatin modification.
Nucleic acid molecules of the instant disclosure can be synthesized by any synthesis method known in the art such as enzymatic synthesis or solid phase synthesis. In one aspect, a dsRNA molecule or directional trigger as disclosed herein is chemically synthesized. In another aspect, a dsRNA molecule or directional trigger as disclosed herein is enzymatically produced. In a further aspect, a dsRNA molecule or directional trigger as disclosed herein is enzymatically produced in vitro. The actual synthesis of the polynucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, "Molecular Cloning: A Laboratory Manual," Sambrook et al., (1989); "Current Protocols in Molecular Biology," Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York (1988) and "Oligonucleotide Synthesis," Gait, M. J., ed. (1984) utilizing solid phase chemistry, e.g,. cyanoethyl phosphoramidite followed by deprotection, desalting and purification by for example, an automated trityl-on method or HPLC. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed, for example, kits from Ambion have DNA ligated on the 5' end that encodes a bacterial T7 polymerase promoter that makes RNA strands that can be assembled into a dsRNA. Alternatively, dsRNA molecules can be produced from expression cassettes in bacterial cells that have regulated or deficient RNase III enzyme activity. In one aspect, design parameters such as Reynolds score and Tuschl rules are known in the art and are used in selecting polynucleotide sequences effective in gene silencing. In another aspect, random design or empirical selection of polynucleotide sequences is used in selecting polynucleotide sequences effective in gene silencing. In a further aspect, the sequence of a polynucleotide is screened against the genomic DNA of the intended plant to minimize unintentional silencing of other genes.
Following synthesis, nucleic acid molecules of the present disclosure may optionally be purified. For example, polynucleotides can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, polynucleotides may be used with no, or a minimum of, purification to avoid losses due to sample processing. The polynucleotides may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to promote annealing and/or stabilization of the duplex strands.
The instant disclosure provides a recombinant construct comprising a plant expressible promoter operably linked to a nucleotide sequence encoding a directional trigger as described herein. In one embodiment, a promoter used herein is selected from the group consisting of a constitutive promoter, a tissue-specific promoter, and an inducible promoter. In one embodiment, a constitutive promoter is the CaMV 35S promoter. In another embodiment, a promoter is an abiotic stress inducible promoter.
The instant disclosure also provides a transgenic plant expressing a directional trigger from the recombinant construct described above. In one embodiment, the directional trigger is stably expressed in the transgenic plant. In another embodiment, the directional trigger is transiently expressed in the transgenic plant.
A dsRNA molecule or directional trigger of the instant disclosure can comprise various chemical modifications including, but not limited to, modified bases, modified sugar backbone, and modified internucleoside linkages. In one aspect, a dsRNA molecule or directional trigger as disclosed herein is chemically modified which modification is capable of enhancing the delivery of the dsRNA molecule or directional trigger into a plant cell, improving its stability in a plant cell, or both. In a further aspect, a dsRNA molecule or directional trigger comprises a cholesterol moiety. In one aspect, a dsRNA molecule or directional trigger as disclosed herein is a combination of ribonucleotides and deoxyribonucleotides, e.g., synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides.
In one aspect, a dsRNA molecule or directional trigger as disclosed herein comprises one or more modified nucleotides of any kind in any part of the directional trigger, preferentially, in a 5' or 3' overhang. Exemplary modified RNA nucleotides can be found in Limbach et al. Summary: the modified nucleosides of RNA. Nucleic Acids Res. 1994, 22(12):2183-96; and Abeydeera et al. 2008, Modified Nucleosides in RNA. Wiley Encyclopedia of Chemical Biology. 1-14. Further exemplary modified nucleotides can comprise a modified base including, but not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3- deazaadenine. In another aspect, a dsRNA molecule or directional trigger as disclosed herein includes a non-canonical nucleotide such as inosine, thiouridine, or pseudouridine.
In another aspect, a dsRNA molecule or directional trigger as disclosed herein comprises a modified polynucleotide backbone including, but not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates.
In another aspect, a dsRNA molecule or directional trigger as disclosed herein is one active ingredient of a herbicidal, insecticidal, or pesticidal composition. A dsRNA molecule or directional trigger of the instant disclosure can be part of a composition further comprising various molecules or agents. In one aspect, a dsRNA molecule or directional trigger as disclosed herein is formulated with counter-ions or other molecules that are known to associate with nucleic acid molecules, e.g., tetraalkyl ammonium ions, trialkyl ammonium ions, sulfonium ions, lithium ions, and polyamines such as spermine, spermidine, or putrescine. In another aspect, a dsRNA molecule or directional trigger as disclosed herein is formulated with a non-polynucleotide herbicide (e.g., glyphosate, 2,4-dichloropropionic acid, bromoxynil, sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates, phthalide, bialaphos, phosphinothricin, glufosinate, atrazine, dicamba, cyclohexanedione (sethoxydim), and aryloxyphenoxypropionate (haloxyfop)). In a further aspect, a dsRNA molecule or directional trigger as disclosed herein constitutes an active ingredient of a liquid herbicidal composition.
In a further aspect, a dsRNA molecule or directional trigger as disclosed herein is formulated with a transferring agent or permeability-enhancing agent which conditions the surface of a plant tissue, e.g., seed, leaves, stems, roots, flowers, or fruits, for permeation by the dsRNA molecule or directional trigger into plant cells. The transfer of a dsRNA molecule or directional trigger as disclosed herein into plant cells can be facilitated by the prior or contemporaneous application of a transferring agent to the plant tissue. The transferring agent enables a pathway for a dsRNA through cuticle wax barriers, stomata and/or cell wall or membrane barriers and into plant cells.
Suitable agents to facilitate transfer of a dsRNA molecule or directional trigger into a plant cell include agents that increase permeability of the exterior of the plant or that increase permeability of plant cells to oligonucleotides or polynucleotides. Such agents include, but are not limited to, a chemical agent, a physical agent, or combinations thereof. Chemical agents for conditioning includes, but are not limited to, (a) surfactants, (b) an organic solvents or an aqueous solutions or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or combinations thereof. A transferring agent contemplated herein can further comprise a humectant or a chelating agent.
Exemplary agents or treatments for conditioning a plant for permeation include, but are not limited to, emulsions, reverse emulsions, liposomes, and other micellar-like compositions. Further exemplary agents or treatments include counter-ions or other molecules that are known to associate with nucleic acid molecules, e.g., inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations. Organic solvents useful in conditioning a plant to permeation by polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, other solvents miscible with water or that will dissolve phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions). Naturally derived or synthetic oils with or without surfactants or emulsifiers can be used, e.g., plant-sourced oils, crop oils, paraffinic oils, polyol-fatty acid esters, and oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine. A composition comprising a dsRNA molecule or directional trigger as disclosed herein can further comprise an organic or inorganic salt. In one aspect the salt is an ammonium salt, for example, ammonium sulfate.
Exemplary surfactants which facilitate the uptake of a dsRNA into plant cells include sodium or lithium salts of fatty acids (such as tallow or tallowamines or phospholipids) and organosilicone surfactants. Further exemplary surfactants include organosilicone surfactants including nonionic organosilicone surfactants, e.g., trisiloxane ethoxylate surfactants or a silicone polyether copolymer such as a copolymer of polyalkylene oxide modified heptamethyl trisiloxane and allyloxypolypropylene glycol methylether (commercially available as Silwet L-77 surfactant). When Silwet L-77 surfactant is used to treat plant seed, leaves or other surfaces, concentrations in the range of about 0.015 to about 2% by weight (wt %) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 wt %) are efficacious in preparing a seed, leaf or other plant surface for transfer of a dsRNA molecule or directional trigger into plant cells.
Exemplary physical agents facilitating the uptake of a dsRNA into plant cells include, but are not limited to, (a) abrasives such as carborundum, corundum, sand, calcite, pumice, garnet, and the like, (b) nanoparticles such as carbon nanotubes, or (c) a physical force. Carbon nanotubes are disclosed by Kam et al. (2004) J. Am. Chem. Soc., 126 (22):6850-6851, Liu et al. (2009) Nano Lett. ,9 (3): 1007-1010 and Khodakovskaya et al. (2009) ACS Nano, 3(10):3221-3227. Physical force agents can include heating, chilling, the application of positive pressure, or ultrasound treatment.
In another aspect, a dsRNA molecule or directional trigger as disclosed herein can be functionally associated with a cell-penetrating peptide which is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (e.g., non-endocytotic) translocation properties associated with transport of the membrane-permeable complex across the plasma and/or nuclear membranes of a cell. Cell-penetrating peptides used in the membrane-permeable complex of the present disclosure preferably comprise at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with a dsRNA that has been modified for such linkage. Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185 . Cell-penetrating peptides of the present disclosure preferably include, but are not limited to, penetratin, transportan, pIsl, TAT(48-60), pVEC, MTS, and MAP.
A dsRNA molecule or directional trigger or a composition comprising a dsRNA molecule or directional trigger of the instant disclosure can be applied to a plant or plant part by any method known in the art, e.g., spraying, drenching, soaking, or coating with a powder, emulsion, suspension, or solution. In one aspect, a dsRNA molecule or directional trigger as disclosed herein is exogenous to a plant cell.
The instant disclosure also provides plants and parts thereof treated with a dsRNA molecule or directional trigger as disclosed herein. The instant disclosure further provides plants and parts thereof comprising a dsRNA molecule or directional trigger as disclosed herein.
In one aspect, plants and parts thereof treated with a dsRNA molecule of the instant disclosure comprise reduced expression of at least 1, 2, or 3 target genes of the dsRNA molecule. In another aspect, plants and parts thereof treated with a dsRNA molecule of the instant disclosure comprise a partial reduction of the expression of at least 1, 2, or 3 target genes of the dsRNA molecule. In a further aspect, plants and parts thereof treated with a dsRNA molecule of the instant disclosure comprise a substantial reduction of the expression of at least 1, 2, or 3 target genes of the dsRNA molecule. In a further aspect, plants and parts thereof treated with a dsRNA molecule of the instant disclosure comprise an effective elimination of the expression of at least 1, 2, or 3 target genes of the dsRNA molecule.
In one aspect, plants and parts thereof comprising a dsRNA molecule of the instant disclosure comprise reduced expression of at least 1, 2, or 3 target genes of the dsRNA molecule. In another aspect, plants and parts thereof comprising a dsRNA molecule of the instant disclosure comprise a partial reduction of the expression of at least 1, 2, or 3 target genes of the dsRNA molecule. In a further aspect, plants and parts thereof comprising a dsRNA molecule of the instant disclosure comprise a substantial reduction of the expression of at least 1, 2, or 3 target genes of the dsRNA molecule. In a further aspect, plants and parts thereof comprising a dsRNA molecule of the instant disclosure comprise an effective elimination of the expression of at least 1, 2, or 3 target genes of the dsRNA molecule.
Any commercially or scientifically valuable plant is envisaged in accordance with some embodiments of the disclosure. Plants that are particularly useful in the methods of the disclosure include all plants which belong to the super family Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia vi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fïmbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize, wheat, barley, rye, oat, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. Alternatively algae and other non-Viridiplantae can be used for the methods of the present disclosure.
According to some embodiments of the disclosure, the plant used by the method of the disclosure is a crop plant including, but not limited to, cotton, Brassica vegetables, oilseed rape, sesame, olive tree, palm oil, banana, wheat, corn or maize, barley, alfalfa, peanuts, sunflowers, rice, oats, sugarcane, soybean, turf grasses, barley, rye, sorghum, sugar cane, chicory, lettuce, tomato, zucchini, bell pepper, eggplant, cucumber, melon, watermelon, beans, hibiscus, okra, apple, rose, strawberry, chili, garlic, pea, lentil, canola, mums, Arabidopsis, broccoli, cabbage, beet, quinoa, spinach, squash, onion, leek, tobacco, potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, and also plants used in horticulture, floriculture or forestry, such as, but not limited to, poplar, fir, eucalyptus, pine, an ornamental plant, a perennial grass and a forage crop, coniferous plants, moss, algae, as well as other plants available on the internet at, for example, nationmaster.com/encyclopedia/Plantae.
According to a specific embodiment, the plant is selected from the group consisting of corn, rice, wheat, tomato, cotton and sorghum. In certain embodiments, the plant is a corn plant. In certain embodiments, the plant is a rice plant. In certain embodiments, the plant is a wheat plant. In certain embodiments, the plant is a cotton plant. In certain embodiments, the plant is a sorghum plant.
Introduction of the compositions of the present disclosure can be performed to any organs/cells of the plant (as opposed to seeds) using conventional delivery methods such as particle bombardment, grafting, soaking and the like.
In one aspect, the instant disclosure also provides a plant seed treated with a composition comprising a dsRNA molecule or directional trigger as disclosed herein. In another aspect, the instant disclosure also provides a plant seed comprising a dsRNA molecule or directional trigger as disclosed herein.
In one aspect, a directional trigger-comprising plant or seed as disclosed herein comprises one or more enhanced traits. As used herein an "enhanced trait" means a characteristic of a transgenic plant that includes, but is not limited to, an enhance agronomic trait characterized by enhanced plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. In more specific aspects of this disclosure an enhanced trait is selected from group consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. In an important aspect of the disclosure the enhanced trait is enhanced yield including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. "Yield" can be affected by many properties including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Yield can also be affected by efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill.
Increased yield of a transgenic or non-transgenic plant of the present disclosure can be measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e. seeds, or weight of seeds, per acre), bushels per acre, tons per acre, or kilo per hectare. For example, corn yield may be measured as production of shelled corn kernels per unit of production area, for example in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, for example at 15.5 percent moisture. Increased yield may result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Nucleic acid molecules as disclosed herein can also be used to provide plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways. Also of interest is the generation of transgenic or non-transgenic plants that demonstrate enhanced yield with respect to a seed component that may or may not correspond to an increase in overall plant yield; such properties include enhancements in seed oil, seed molecules such as protein and starch, oil components as may be manifest by an alterations in the ratios of seed components.
The instant disclosure further provides a container of plant seeds treated with a dsRNA molecule or directional trigger as disclosed herein. A container of treated seeds of the instant disclosure may contain any number, weight, or volume of seeds. For example, a container can contain at least, or greater than, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, the container can contain at least, or greater than, 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more seeds. Containers of seeds may be any container available in the art. By way of non-limiting example, a container may be a box, a bag, a packet, a pouch, a tape roll, or a tube.
The instant disclosure provides a method of regulating the expression of two or more genes of interest simultaneously by introducing into a plant cell a dsRNA molecule or directional trigger as disclosed herein. Also provided is a method of disrupting a metabolic pathway by introducing into a plant cell a dsRNA molecule or directional trigger as disclosed herein which targets two or more genes of interest in the metabolic pathway. Further provided is a method of simultaneously regulating the expression of two or more genes of interest selected from the group consisting of a plant endogenous gene sequence, a plant phytopathogen gene sequence, a plant viral gene sequence, a plant insect gene sequence, and combinations thereof, wherein the method comprises introducing into a plant cell a dsRNA molecule or directional trigger as disclosed herein.
The instant disclosure also provides a method of increasing the homogeneity or uniformity of a sRNA population processed from a dsRNA molecule by a Dicer-like protein, wherein the method comprises introducing to a dsRNA molecule one, two, three, four, five or more features selected from the group consisting of (a) having a length between about 45 and about 75 nucleotides, (b) comprising one or more Adenine- or Uracil-rich linker sequences adjoining the two or more sRNA trigger sequences each of which encodes a sRNA duplex, (c) comprising a 3' overhang in the antisense strand, (d) comprising a Uracil at positions 20 and 21 in the antisense strand relative to terminus of the 3' overhang, (e) a 5' overhang of 5 nucleotides long, and (f) combinations thereof.
The instant disclosure also provides a method of enriching functional sRNAs processed from a trigger molecule, comprising introducing into a trigger molecule one, two, three, four, five or more features selected from the group consisting of (a) two or more sRNA trigger sequences each of which encodes a sRNA, where the two or more sRNA trigger sequences are not found in a single naturally occurring molecule or not contiguous in a single naturally occurring molecule, (b) having a length between about 45 and about 75 nucleotides, (c) comprising one or more Adenine- or Uracil-rich linker sequences adjoining the two or more sRNA trigger sequences, (d) comprising a 3' overhang in the antisense strand, (e) comprising a Uracil at positions 20 and 21 in the antisense strand relative to terminus of the 3' overhang, (f) a 5' overhang of 5 nucleotides, and (g) combinations thereof.
The instant disclosure also provides a method of producing a trigger molecule with pre-determined processing pattern, comprising introducing into a trigger molecule one, two, three, four, five or more features selected from the group consisting of (a) two or more sRNA trigger sequences each of which encodes a sRNA, where the two or more sRNA trigger sequences are not found in a single naturally occurring molecule or not contiguous in a single naturally occurring molecule, (b) having a length between about 45 and about 75 nucleotides, (c) comprising one or more Adenine- or Uracil-rich linker sequences adjoining the two or more sRNA trigger sequences, (d) comprising a 3' overhang in the antisense strand, (e) comprising a Uracil at positions 20 and 21 in the antisense strand relative to terminus of the 3' overhang, (f) a 5' overhang of 5 nucleotides, and (g) combinations thereof.
It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, may also be provided in combination in a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, may also be provided separately or in any suitable subcombination or as suitable in any other described aspect of the disclosure. Certain features described in the context of various aspects are not to be considered essential features of those aspects, unless the aspect is inoperative without those elements. Various aspects and aspects of the present disclosure as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
The following Examples are presented for the purposes of illustration and should not be construed as limitations.

EXAMPLES

Example 1: Evaluating the influence of dsRNA overhangs over dsRNA processing and its ability to induce silencing.

Six dsRNA trigger molecules (SEQ ID NO: 1/SEQ ID NO:57, SEQ ID NO:2/SEQ ID NO:57, SEQ ID NO:3/SEQ ID NO:57, SEQ ID NO:/4SEQ ID NO:57, SEQ ID NO:5/SEQ ID NO:57, and SEQ ID NO:6/SEQ ID NO:57), each of which comprises two strands of ∼50 nucleotides with overhangs of various lengths, were incubated in wheat germ extract to test effects of overhang lengths over dsRNA processing. These dsRNA trigger molecules comprise target-specific sequences from a tomato (Solanum lycopersicum, Sl) 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. All trigger molecules are 5'-end-labeled with P32 ( FIG. 1A ). Trigger 1 (SEQ ID NO: 1/SEQ ID NO:57) has blunt ends. Trigger 2 (SEQ ID NO:2/SEQ ID NO:57) has 2-base 3' overhangs on both ends. Triggers 3-6 (SEQ ID NO:3/SEQ ID NO:57, SEQ ID NO:/4SEQ ID NO:57, SEQ ID NO:5/SEQ ID NO:57, and SEQ ID NO:6/SEQ ID NO:57) have on each end 2-base, 5-base, 10-base and 15-base 5'overhangs, respectively.
Specifically, 1 µl of 5' P32-end-labeled dsRNA trigger molecule (50,000 cpm) was incubated in a 40-µl reaction containing 30 µl of wheat germ extract (Promega), and 8 µl 5x Dicer reaction buffer (0.5 M NaCl, 100 µM GTP, 500 µM ATP, 10 mM creatine phosphate, 10 µg/ml creatine phosphokinase, 5 mM DTT, and 0.1 U/µl RNasin) (Promega) at 25°C for 3 h. Reactions were stopped by the addition of 2x proteinase K buffer (200 mM Tris-HCl at pH 7.5, 25mM EDTA, 300mM NaCl, 2% (w/v) sodium dodecyl sulfate) followed by deproteinization with 2 mg/ml proteinase K at 65°C for 15 min. Processed RNA products were precipitated with 3 volumes of cold ethanol and analyzed by electrophoresis in a 15% polyacrylamide sequencing gel. Both Triggers 1 and 2 were processed into small RNAs of 20∼21 nucleotides, while almost no processed small RNA was observed from Triggers 3 to 6, supporting that 5' overhangs delay or prevent the processing of a dsRNA molecule ( FIG. 1B ).
Triggers 1 to 6 were further tested in Nicotiana benthamiana protoplasts for its ability to silence an EPSPS gene. Specifically, 3 µg of each of Triggers 1 to 6 were added to Nicotiana benthamiana protoplasts. Expression of the target EPSPS gene was quantified using Taqman quantitative PCR. Various extents of EPSPS down-regulation were observed from Triggers 1 to 6 ( FIG. 1C ). The degree of EPSPS silencing by triggers having 5' overhangs on both ends decreases as the 5' overhang length increases.

Example 2: Rational design of a directional trigger.

To improve the silencing efficiency of a dsRNA molecule, various sequence or structural features were incorporated into a dsRNA molecule to form a directional trigger. These sequence or structural features facilitate processing a dsRNA following a more predictable pattern and thereby allow for a more focused production of small RNAs functional in silencing intended target genes ( FIG. 2 to FIG. 5 ). Structural features include, without limitation, directional initiation of processing by a Dicer-like protein from one end with a 3' overhang, and use of a 5' overhang in the opposite end blocking initiation of processing by Dicer-like proteins from that end.
A dsRNA molecule synthesized by a RNA dependent RNA polymerase (RDR) (FIG. 2A, RDR6 shown as an example) comprises 3' overhangs on both ends, and therefore is processed by a Dicer-like protein at similar frequencies starting from either end. In contrast, an exemplary directional trigger comprises on its antisense strand a 2-nt 3' overhang ("3' initiator overhang") and a 5' overhang ("5' blocker overhang") which, without being bound to any scientific theory or mechanism, favors and disfavors the initiation of dicer processing from that end, respectively ( FIG. 2B ). Accordingly, a rationally designed directional trigger predominantly produces siRNA products that are in a pre-programmed and predictable phase (e.g., a phase interval of ∼21 nucleotides starting from the dsRNA end with a 3' overhang).
A pre-programmed and predictable processing pattern also allows a dsRNA molecule to give rise to multiple distinct siRNAs in proper phases so that each of the siRNAs is functional in promoting the silencing of its intended target. For example, an exemplary directional chimeric trigger predominantly produces siRNA products that are in a proper phase to target two genes of interest (GOI1 and GOI2) ( FIG. 3 ). Meanwhile, the processing of a directional dsRNA trigger leads to only a minor fraction of its siRNA products with an aberrant phase ( FIG. 3 ). Aberrantly phased siRNA products have at best a partial complementarity with a target sequence which are therefore not capable of promoting target silencing (e.g., non-functional siRNAs in FIG. 3 ).
Additional features were also incorporated into a directional trigger, for example, a AU-rich linker between two target-specific sequences ( FIG. 4 and FIG. 5 ). An exemplary directional trigger ( FIG. 4 ) can be processed into two distinct siRNAs recognizing sequences in the same or different target genes (GOI1 only, or GOI1 and GOI2). Both siRNA1 and siRNA2 comprise a UU di-nucleotide at the 5' end of the antisense strand and a G at the 5' end of the sense strand. Antisense strands of siRNA1 and siRNA2 starting with a UU di-nucleotide are preferentially loaded into Argonaute proteins (AGO) and recognize target gene mRNA molecules leading to target gene silencing. Target-specific sequences are sequences that are essentially identical, identical, or essentially complementary, or complementary, to a sequence of a target gene. Non-specific sequences are sequences that are not related to a target gene and can vary in both length and composition from those shown in FIG. 4 .
A further schematic comparison between an exemplary directional dsRNA trigger and a non-directional dsRNA trigger is shown in FIG. 5 . An exemplary directional dsRNA trigger comprises target-specific sequences from genes of interest (GOIs) on its sense strand. The antisense strand of the exemplified directional dsRNA trigger comprises both a 3' overhang (2-nt exemplified) and a 5' overhang (3-5 nucleotides or longer). A Dicer-like protein cleaves a first 21-24mer (siRNA1) from a directional dsRNA trigger preferentially starting from the end with a 3' overhang, and continues to produce a second 21-24mer (siRNA2) which is immediately next to the first 21-24mer (e.g., in phase with the first 21-24mer). Accordingly, the exemplified directional dsRNA trigger produces a collection of 21-24mers (two 21-24mers are shown in the figure) in a phased manner with siRNA1 and siRNA2 being the predominant species. Further, both siRNA1 and siRNA2 comprise a UU di-nucleotide at the 5' end of their antisense strands and a G at the 5' end of their sense strands. Antisense strands of siRNA1 and siRNA2 starting with a UU di-nucleotide are preferentially loaded into Argonaute proteins (AGO), and are also called guide strands which guide the recognition of target gene mRNA sequences and lead to target gene silencing.
A non-directional dsRNA trigger, however, has no directionality bias towards either end of the trigger when processed by a dicer-like protein. Accordingly, 21-24mers produced from a non-directional dsRNA trigger are more heterogeneous. In-phase 21-24mers (e.g., siRNA1' and siRNA2') represent only a fraction of the total pool of 21-24mers which also comprise substantial out-of-phase 21-24mers (e.g., siRNA3' and siRNA4'). Accordingly, a non-directional dsRNA trigger produces more diluted in-phase 21-24mers compared to a directional trigger. A non-directional dsRNA trigger can be chimeric or non-chimeric, blunt-ended, having 3' overhangs on both ends, or a combination of these features. A chimeric trigger having two 3' overhangs is shown in FIG. 5B .
Further, 21-24mers produced from a non-directional dsRNA trigger lack a UU di-nucleotide at the 5' end of their antisense strand and a G at the 5' end of their sense strand. Accordingly, neither the antisense strand, nor the sense strand is preferentially loaded into a AGO protein. Instead, each strand of every 21-24mer can potentially be loaded into an AGO protein as a guide strand. However, only guide strands 1' and 2' are complementary to target sequences and capable of recognizing target molecules to cause silencing. Therefore, a non-directional dsRNA trigger produces more diluted guide strands that are effective in causing silencing compared to guide strands produced from a directional dsRNA trigger.

Example 3: Evaluating silencing efficiencies of directional dsRNA trigger using a luciferase reporter system in wheat germ extract.

Exemplary directional dsRNA triggers were tested in wheat germ extract for their abilities to silence a luciferase reporter. Three directional dsRNA triggers were used (SEQ ID NO:7/SEQ ID NO:58, SEQ ID NO:8/SEQ ID NO:59, SEQ ID NO:9/SEQ ID NO:60, FIG. 6A ). Each directional dsRNA trigger comprises two target-specific sequences. SEQ ID NO:7/SEQ ID NO:58 comprises one target-specific sequence from AtEPSPS and the other from AtCUT1. Both target-specific sequences in SEQ ID NO:8/SEQ ID NO:59 are from AtEPSPS, whereas both target-specific sequences in SEQ ID NO:9/SEQ ID NO:60 are from AtCUT1. A non-directional dsRNA trigger specifically targeting AtEPSPS (SEQ ID NO:10/SEQ ID NO:61) was also included as a control.
To evaluate silencing activities in wheat germ extract, a 50 µl reaction system was used which contained 25 µl wheat germ extract, 4 µl amino acid mix, a trigger (60 pmoles), and a fusion mRNA comprising a target of the trigger and firefly luciferase (3 pmoles). Reactions were incubated at 25°C for 2 hours after which luciferase activities were read using the dual luciferase system (Promega) according to the manufacturer instructions.
Relative luciferase activities showed successful silencing of a fusion target mRNA AtEPSPS1:Fluc by SEQ ID NO:7/SEQ ID NO:58 and SEQ ID NO:8/SEQ ID NO:59 in wheat germ extract ( Figure 6B ). AtEPSPS1:Fluc is an mRNA fusion between a full-length luciferase coding sequence and an AtEPSPS1 coding sequence which is targeted by directional triggers SEQ ID NO:7/SEQ ID NO:58, SEQ ID NO:8/SEQ ID NO:59, and non-directional trigger SEQ ID NO:10/SEQ ID NO:61. Relative luciferase activities also showed silencing of a fusion target mRNA AtCUT1:Fluc by SEQ ID NO:7/SEQ ID NO:58 and SEQ ID NO:9/SEQ ID NO:60, but not SEQ ID NO:8/SEQ ID NO:59 in wheat germ extract ( FIG. 6C ). AtCUT1:Fluc is an mRNA fusion between a full-length luciferase coding sequence and an AtCUT1 coding sequence that is targeted by triggers SEQ ID NO:7/SEQ ID NO:58 and SEQ ID NO:9/SEQ ID NO:60, but not SEQ ID NO:8/SEQ ID NO:59.

Example 4: Silencing of an endogenous AtEPSPS1 gene in Arabidopsis protoplasts by directional dsRNA triggers.

Exemplary directional dsRNA triggers (SEQ ID NO:7/SEQ ID NO:58, SEQ ID NO:8/SEQ ID NO:59, SEQ ID NO:9/SEQ ID NO:60, FIG. 6A ) were also tested in Arabidopsis protoplasts for their abilities to silence an endogenous AtEPSPS1 gene. A non-directional dsRNA trigger (SEQ ID NO:10/SEQ ID NO:61) was also included as a control. Preparation and transformation of Arabidopsis protoplasts and subsequent RNA extraction are described below in Example 5. SEQ ID NO:7/SEQ ID NO:58, SEQ ID NO:9/SEQ ID NO:60 and SEQ ID NO:10/SEQ ID NO:61 were all tested at a dosage of 200 pmol, while SEQ ID NO:8/SEQ ID NO:59, which contains two distinct siRNAs both targeting AtEPSPS1, was evaluated at a 50 pmol dosage. AtEPSPS1 expression was quantified by q-PCR.
Directional dsRNA triggers containing at least one target-specific sequence from AtEPSPS1 (SEQ ID NO:7/SEQ ID NO:58 and SEQ ID NO:8/SEQ ID NO:59) were capable of specifically down regulating AtEPSPS1 expression, while the directional dsRNA trigger only comprising target-specific sequences from AtCUT1 (SEQ ID NO:9/SEQ ID NO:60) could not (FIG. 6D). The lower dosage used for SEQ ID NO:8/SEQ ID NO:59 (50 pmol) which achieved a level of target down-regulation comparable to that by a higher dosage of trigger SEQ ID NO:7/SEQ ID NO:58 (200 pmol) shows that by having two target sequences from AtEPSPS1, trigger SEQ ID NO:8/SEQ ID NO:59 is more efficient in promoting silencing compared to trigger SEQ ID NO:7/SEQ ID NO:58 (having only one target sequence from AtEPSPS1). Both AtCUT1 (At1g68530) and AtEPSPS1 (At1g48860) genes are expressed in Arabidopsis leaves, which are sources for protoplast preparation.

Example 5: Isolation and PEG-mediated transformation of Arabidopsis protoplasts and subsequent RNA extraction.

Isolation and PEG-mediated transformation of Arabidopsis protoplasts were conducted following standard protocols. In short, dark green, healthy Arabidopsis leaves were collected from about 3 week old plants prior to bolting. Leaves were then sliced on a piece of parafilm into approximately ∼1 mm strips starting from the tip of a leaf and ending at ∼2 to 3 mm from the petiole end of a leaf. Sliced leaves were then placed into a digestion solution in a Petri dishes (∼50 leaves per Petri dish) with the adaxial leaf surface facing up. Leaf stripes were infiltrated with the digestion solution by the application of vacuum followed by an overnight incubation at room temperature in dark. Protoplasts were then released from leaves into the digestion solution after a gentle shaking of the Petri dish for 2-3 minutes at 40 RPM. Subsequently, the digestion mixture was filtered through two layers of 60-micron nylon mesh into a 50 ml conical tube. The Petri dish and leaf strips were rinsed by a 10 ml W5 solution which was subsequently filtered with collected. After a gentle and thorough mixing of protoplasts with the W5 rinsing solution, protoplasts were spun down in a Harrier benchtop centrifuge with a swinging bucket rotor at 100 x g for 2 minutes. A protoplast pellet was resuspended in 10 ml W5 solution. Protoplasts collected from multiple Petri dishes were then pooled with protoplast concentration estimated using a hemacytometer (40 leaves generally give about 4-6 x 10⁶ protoplasts). Prepared protoplasts were left in W5 solution on ice for at least 1 hour before transformation.
For analyzing the silencing of a reporter gene by a dsRNA molecule, 0.8 to 1 x 10⁵ Arabidopsis protoplasts were used per transformation. For each transformation, 10 µg of each reporter construct together with 1.3 µg FLuc (pMON8796) a (pMON63934) as internal controls was used. For each construct, triplicate samples were used with a randomized transformation order. DNA constructs were firstly mixed with 150 µl protoplasts by gentle pipetting up and down. Subsequently, a 150 µl PEG solution was added into the protoplasts followed by mixing of the protoplast and PEG solutions by inverting the tube for about 1 minute. After a 4-minute incubation at room temperature, the PEG-protoplast transformation mixture was mixed with a 300 µl W5 solution followed by an incubation for 5-10 minutes. Protoplasts were then spun down by a centrifugation at 90 x g for 1 minute, and subsequently resuspended in 1 ml WI solution. Transformed protoplasts were incubated in dark at room temperature for 4-6 hours prior to RNA extraction and Taqman or Transcriptional profiling analysis.
For RNA extraction, approximately 1 x 10⁶ Arabidopsis protoplasts were spun down at 300 x g for 2 minutes. The protoplast pellet was resuspended and lysed in 250 µl Buffer RLT (Qiagen RNeasy Mini Kit or Qiagen # 79216) containing 1% β-Mercaptoethanol (BME). Protoplasts lysed by Buffer RLT were either stored in a -80°C freezer or processed immediately for RNA isolation according to the standard plant/fungi extraction protocol found in Qiagen's RNeasy Mini Kit.

Example 6: Silencing of tomato or tobacco target genes by directional dsRNA triggers.

Exemplary directional dsRNA triggers having tomato target genes were tested in wheat germ extract for their abilities to silence a luciferase reporter. Two directional dsRNA triggers were tested (SEQ ID NO:11/SEQ ID NO:62, SEQ ID NO:12/SEQ ID NO:63, FIG. 7A ). SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63 each comprise two target sequences, one from (Solanum lycopersicum, Sl) phytoene desaturase (SlPDS) and the other from SlEPSPS. Arrangements of the two target sequences are reversed between SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63. SEQ ID NO:9/SEQ ID NO:60 was also included as a control, which comprises two target-specific sequences from AtCUT1. Processing of dsRNA molecules in wheat germ extract and monitoring of the luciferase reporter activity were performed essentially as described in Example 3.
Relative luciferase activities showed successful silencing of a fusion target mRNA SlPDS:Fluc by SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63 in wheat germ extract ( FIG. 7B ). SlPDS:Fluc is an mRNA fusion between a full-length luciferase coding sequence and a SlPDS coding sequence which is targeted by SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63, but not SEQ ID NO:9/SEQ ID NO:60. Relative luciferase activities also showed silencing of a fusion target mRNA SlEPSPS:Fluc by SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63, but not SEQ ID NO:9/SEQ ID NO:60, in wheat germ extract ( FIG. 7C ). SlEPSPS:Fluc is an mRNA fusion between a full-length luciferase coding sequence and a SlEPSPS coding sequence which is targeted by triggers SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63, but not SEQ ID NO:9/SEQ ID NO:60.
Directional dsRNA triggers SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63 were further tested in Nicotiana benthamiana (Nb) protoplasts for their abilities to silence an endogenous NbEPSPS1 gene. NbEPSPS1 expression was evaluated by Northern blots following treating Nicotiana benthamiana protoplasts with SEQ ID NO:11/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63 ( FIG. 7D ). Quantification of NbEPSPS1 expression via either a 5' probe or a 3' probe indicates that both SEQ ID NO:1 1/SEQ ID NO:62 and SEQ ID NO:12/SEQ ID NO:63 are capable of silencing NbEPSPS1 in Nicotiana benthamiana protoplasts ( FIG. 7E ).

Example 7: A comparison between a directional dsRNA trigger and a non-directional trigger

A directional dsRNA trigger (SEQ ID NO:8/SEQ ID NO:59 targeting AtEPSPS1, see FIG. 6A ) and a non-directional trigger (SEQ ID NO:10/SEQ ID NO:61 targeting AtEPSPS1) were both tested in Arabidopsis protoplasts to compare their efficiencies in promoting the silencing of an endogenous AtEPSPS1 gene. Arabidopsis protoplast preparation and transformation were carried out essentially as described in Examples 4 and 5. Eight different dsRNA dosages (250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, and 2.0 pmol) were tested. Directional triggers SEQ ID NO:14/SEQ ID NO:64 and SEQ ID NO:14/SEQ ID NO:81 which do not target AtEPSPS1 were also included as negative controls.

AtEPSPS1 expression was quantified using q-PCR with statistic significance evaluated by Student's t-test. AtEPSPS1 expression data showed that directional dsRNA trigger SEQ ID NO:8/SEQ ID NO:59 had a higher silencing efficiency compared to non-directional trigger SEQ ID NO:10/SEQ ID NO:61 ( FIG. 8 and Table 1). SEQ ID NO:8/SEQ ID NO:59 reduced AtEPSPS1 expression by 34% and 39% when used at 125 and 250 pmol, respectively. SEQ ID NO:10/SEQ ID NO:61, however, was able to reduce AtEPSPS1 expression by 24% at the highest dose (250 pmol), and showed no silencing activity when used at a concentration of 125 pmol or lower. When used at the same concentration (e.g., 250 pmol), directional dsRNA trigger SEQ ID NO:8/SEQ ID NO:59 is more effective in reducing AtEPSPS1 expression compared to non-directional trigger SEQ ID NO:10/SEQ ID NO:61 (39% versus 24%). No AtEPSPS1 silencing was observed with the use of negative control triggers SEQ ID NO:14/SEQ ID NO:64 and SEQ ID NO:14/SEQ ID NO:81, demonstrating that the observed AtEPSPS1 silencing is sequence specific and dependent on the presence of a trigger molecule against AtEPSPS1.

Table 1: Quantification results of AtEPSPS1 expression in Arabidopsis protoplasts by q-PCR following treatments with dsRNA triggers at various dosages.

Treatment	Mean SRC	Relative Expression changes
01_250_SEQ ID NO:10/SEQ ID NO:61	75.552	-24%
02_125_SEQ ID NO:10/SEQ ID NO:61	97.548	-2%
03_62.5_SEQ ID NO:10/SEQ ID NO:61	150.617	51%
04_31.3_SEQ ID NO:10/SEQ ID NO:61	113.633	14%
05_15.6_SEQ ID NO:10/SEQ ID NO:61	145.935	46%
06_7.8_SEQ ID NO:10/SEQ ID NO:61	121.434	21%
07_3.90_SEQ ID NO:10/SEQ ID NO:61	121.01	21%
08_2.0_SEQ ID NO:10/SEQ ID NO:61	147.592	48%
09_250_SEQ ID NO:8/SEQ ID NO:59	60.51	-39%
10_125_SEQ ID NO:8/SEQ ID NO:59	65.922	-34%
11_62.5_SEQ ID NO:8/SEQ ID NO:59	93.578	-6%
12_31.3_SEQ ID NO:8/SEQ ID NO:59	105.186	5%
13_15.6_SEQ ID NO:8/SEQ ID NO:59	98.586	-1%
14_7.8_SEQ ID NO:8/SEQ ID NO:59	114.773	15%
15_3.90_SEQ ID NO:8/SEQ ID NO:59	124.326	24%
16_2.0_SEQ ID NO:8/SEQ ID NO:59	128.81	29%
17_250_SEQ ID NO:14/SEQ ID NO:64	145.988	46%
18_125_SEQ ID NO:14/SEQ ID NO:64	119.543	20%
19_62.5_SEQ ID NO:14/SEQ ID NO:64	144.543	45%
20_31.3_SEQ ID NO:14/SEQ ID NO:64	139.518	40%
21_15.6_SEQ ID NO:14/SEQ ID NO:64	136.958	37%
22_7.8_SEQ ID NO:14/SEQ ID NO:64	139.309	39%
23_3.90_SEQ ID NO:14/SEQ ID NO:64	118.386	18%
24_2.0_SEQ ID NO:14/SEQ	135.544	36%
ID NO:64
25_250_SEQ ID NO:14/SEQ ID NO:81	140.861	41%
26_125_SEQ ID NO:14/SEQ ID NO:81	111.536	12%
27_62.5_SEQ ID NO:14/SEQ ID NO:81	131.118	31%
28_31.3_SEQ ID NO:14/SEQ ID NO:81	129.451	29%
29_15.6_SEQ ID NO:14/SEQ ID NO:81	133.988	34%
30_7.8_SEQ ID NO:14/SEQ ID NO:81	147.589	48%
31_3.90_SEQ ID NO:14/SEQ ID NO:81	126.356	26%
32_2.0_SEQ ID NO:14/SEQ ID NO:81	154.903	55%
no trigger	100	baseline

Table 1 shows the same dataset used in FIG. 8 . Each experiment is shown in the Treatment column as "treatment number"_"trigger dosage"_"trigger name." For example, "01_250_SEQ ID NO:10/SEQ ID NO:61" refers to treatment number 01 which uses 250 pmol of trigger SEQ ID NO:10/SEQ ID NO:61. Mean SRC refers to an average readout of AtEPSPS1 expression level normalized to a no-trigger control which is set at 100. The rightmost column refers to the percentage of AtEPSPS1 expression change normalized to a no-trigger control (shown as "baseline" in the bottom row).

Example 8: Analyzing small RNAs processed from a directional dsRNA trigger by deep sequencing.

To confirm that the rational design of a directional dsRNA trigger indeed promotes dsRNA processing in a more predictable manner and enriches for small RNAs functional for silencing an intended target, small RNA deep sequencing was carried out to analyze the processing products of directional dsRNA triggers SEQ ID NO:7/SEQ ID NO:58, SEQ ID NO:8/SEQ ID NO:59, and SEQ ID NO:9/SEQ ID NO:60 (see FIG. 6A ). Directional dsRNA triggers were first processed in wheat germ extract with processed RNA products collected and subject to deep sequencing thereafter. Deep sequencing results are summarized in Table 2. The sequencing results demonstrate that the processing of SEQ ID NO:7/SEQ ID NO:58, SEQ ID NO:8/SEQ ID NO:59, and SEQ ID NO:9/SEQ ID NO:60 into small RNAs of 21-24 nucleotides (21-24mers) is directionally biased towards the 3' end of their antisense strands (AS strand). Specifically, 49%, 51%, and 69% of 21-24mers are mapped to the 3' end of the antisense strand of SEQ ID NO:7/SEQ ID NO:58, SEQ ID NO:8/SEQ ID NO:59, and SEQ ID NO:9/SEQ ID NO:60, respectively.

Table 2. The processing of directional dsRNA triggers SEQ ID NO:7/SEQ ID NO:58, SEQ ID NO:8/SEQ ID NO:59, and SEQ ID NO:9/SEQ ID NO:60 (see FIG. 6A) into 21-24mers is directionally biased towards the 3' end of the antisense strand (AS strand). "S strand" refers to the sense strand of a dsRNA trigger molecule.

Trigger	T number	Targets	Total reads (all sizes)	Total reads (21-24 mer)	21-24 from 3' of AS strand	21-24 from 5' of S strand	% of 21-24 from 3' AS strand	% of 21-24 from 3' S strand
1	SEQ ID NO:7/SE Q ID NO:58	AtEPSPS/AtC UT1	1654000	232000	112000	1200	49	0.5
2	SEQ ID NO:8/SE Q ID NO:59	AtEPSPS1	176000	81630	42232	19.793	51	24
3	SEQ ID NO:9/SE Q ID NO:60	AtCUT1	2456774	473000	430000	2000	69	0.4

A closer analysis of the deep sequencing results from directional dsRNA trigger SEQ ID NO:9/SEQ ID NO:60 further illustrates the directionality of SEQ ID NO:9/SEQ ID NO:60 processing and an enrichment of 21-24mers that are functional in silencing SEQ ID NO:9/SEQ ID NO:60's intended target AtCUT1 ( FIG. 9 and FIG. 10 ). FIG. 9B shows the size distribution and relevant abundance of RNA products from SEQ ID NO:9/SEQ ID NO:60 after processing in wheat germ extract. In total, 2,456,774 sequencing reads are mapped to trigger SEQ ID NO:9/SEQ ID NO:60, of which 2,107,001 reads (∼85%) are from the antisense strand of trigger SEQ ID NO:9/SEQ ID NO:60 while 349,774 sequencing reads (∼15%) are from the sense strand of trigger SEQ ID NO:9/SEQ ID NO:60. FIG. 9C shows that the size range of 21-24 nucleotides (21-24mer) contains 473,000 sequencing reads (∼19% of total sequencing reads), of which ∼90% are mapped to the 3' end of the antisense strand of trigger SEQ ID NO:9/SEQ ID NO:60 with only ∼0.4% from the 5' end of the sense strand. An bias towards the 3' end of the antisense strand compared to the antisense 5' end (∼17% versus ∼0.4%) supports a preferential, directional processing of SEQ ID NO:9/SEQ ID NO:60 starting from the end comprising a 2-nt 3' overhang.
Additional deep sequencing experiments were also conducted to analyze the pattern of directional trigger processing into siRNAs. A 48-nt directional trigger (SEQ ID NO:15, top row in FIG. 11 ) was also processed in wheat germ extract with small RNA products collected and subject to deep sequencing. Deep sequencing results for 21-24mers were analyzed and assembled in silico to identify putative primary siRNA duplexes (e.g., perfect-match double-stranded 21-24mers). These putative siRNA duplexes are ranked based on their relative abundance estimated by the sum of their absolute frequencies. Top ranked putative duplexes (only perfect-match 21-24mers) were aligned against the BOL5.2 48-nt trigger sequence ( FIG. 11 ). The top 10 putative siRNA duplexes amount to ∼75% of all perfect match reads. Among these 10 duplexes, only 3 preferentially match to the 3' side of the trigger (the dsRNA end with a 5' overhang). Four top-ranked duplexes (two 21-nt and two 24-nt siRNAs) showed opposite strand biases. The two top-ranked 21-nt siRNAs are biased towards the antisense strand, while the two top-ranked 24-nt siRNAs have a bias to the sense strand.

Example 9: In planta processing of directional chimeric triggers

A total of 20 µl of 4 µg/µl stock of dsRNA trigger (GFP targeting trigger alone - SEQ ID NO:65/SEQ ID NO:82 or chimeric GFP/MgChl - SEQ ID NO:66/SEQ ID NO:67) was applied to the adaxial side of leaves of Nicotiana benthamiana 16C transgenic plants (2-3 weeks old seedlings) and introduced into the plant cells. The final solution consisted of the dsRNA trigger and water. The solution was left to dry on the surface of the leaf for approximately one hour after which sandpaper was rolled on the leaf to deliver the dsRNA into plant cells.
Plant tissue was assessed for visual phenotype at four days post transfection. At the same time, tissue was harvested for Western Blot analysis. For each treatment two 5 mm leaf discs were placed in a frozen 1.5mL microtube. The tissue was ground with a frozen plastic micropestle until a fine powder was formed. Approximately 20 µl of a buffer solution was added to each sample, followed by vortexing for thirty seconds. The buffer solution consisted of 50mM Tris-HCl, pH 7.4; 2.5 mM MgCl2, 100 mM KCl; 0.1% Nonidet P-40 and one tablet of Complete proteinase inhibitor tablet (Roche). The extract was then centrifuged at maximum speed for 10 minutes at 4°C in a microcentrifuge. The supernatant was transferred to a clean tube and centrifuged for an additional 5 minutes at 4°C. Total protein was quantified using the BCA assay (Pierce). For immunoblot analysis, 6 µg of total protein were analyzed using standard Western blotting procedures. GFP and MgChl were visualized using a 1:5000 dilution of polyclonal anti-GFP (Santa Cruz Biotechnologies) or polyclonal anti-MgChl (produced in house) rabbit antibodies, respectively, as illustrated in FIG. 12 . This treatment was followed by the use of an HRP conjugate (goat anti-rabbit IgG-HRP; Santa Cruz Biotechnologies). Detection of the GFP or MgChl specific bands was performed by using the SuperSignalWest Pico chemiluminescent substrate (Pierce).
Visual examination of the plants at 4 days after treatment revealed suppression of GFP in both treatments, those treated with GFP-only trigger or those treated with the chimeric trigger to GFP and MgChl. However, only the leaves treated with the chimeric trigger targeting both GFP and MgChl displayed the MgChl suppression phenotype characterized by the presence of yellow/chlorotic foci, visible in plain light. Image J software was used to quantify the percentage reduction for both targets by measuring the band intensity after Western blot. The results are presented in Table 3. Target protein was reduced by at least 60%. The percent knockdown established for MgChl was likely underestimated since the tissue used for protein extraction consisted of both green and yellow sectors. Table 3. Percent reduction of corresponding protein targets. Values were calculated based on comparison of band intensity between untreated and trigger-treated samples.

GFP % reduction MgChl % reduction

Untreated 0 0

GFP-only trigger (T41817) 100 0

Chimeric trigger (T52255) 95 60

Example 10. Mutations in the central portion of the directional trigger sequence and blunt ends (at the ends of the trigger) resulted in loss of efficacious processing.

An evaluation of the efficacy of processing was undertaken by introducing mutations (SEQ ID NO:70/SEQ ID NO:71; see FIG. 13 , panel A) within the complementary portion of the dsRNA trigger SEQ ID NO:68/SEQ ID NO:69, thus presumably inhibiting effective processing or dicing of the polynucleotide when introduced in Arabidopsis protoplasts. Additionally, a blunt ended dsRNA trigger (SEQ ID NO:72/SEQ ID NO:73; see FIG. 13 , panel A) was also tested.
Arabidopsis protoplasts were transfected using standard procedures as described in Example 5, with 100 pmol each of dsRNA triggers, including a nonspecific trigger (SEQ ID NO:74/SEQ ID NO:75) as negative control, a directional trigger targeting only Arabidopsis Phytoene Desaturase (PDS) (SEQ ID NO:76/SEQ ID NO:77), a directional trigger targeting only Arabidopsis Phosphoribosylanthranilate transferase 1 (PAT1) (SEQ ID NO:78/SEQ ID NO:79), the directional trigger targeting both PDS and PAT1 (SEQ ID NO:68/SEQ ID NO:69), the mutated directional trigger (SEQ ID NO:70/SEQ ID NO:71), and the blunt ended trigger (SEQ ID NO:72/SEQ ID NO:73). RNA was extracted approximately 16-20 hrs after transfection and analyzed by Taqman. The result of this analysis are presented in FIG. 13 , panel B.
In the analysis conducted on PDS transcript ( FIG. 13 , panel B, left side), a clear reduction of message levels is visible in the cells treated with the dsRNA targeting PDS only (SEQ ID NO: 76/SEQ ID NO:77) or in the cells treated with the chimeric PDS/PAT1 dsRNA trigger (SEQ ID NO:68/SEQ ID NO:69), but not in the cells treated with the mutated trigger (SEQ ID NO:70/SEQ ID NO:71) or the trigger with blunt ends (SEQ ID NO:72/SEQ ID NO:73). Likewise, when Taqman analysis was conducted assaying PAT1 mRNA levels ( FIG. 13 , panel B, right side), a reduction in message was only observed in the cells treated with dsRNA polynucleotides targeting PAT1 (SEQ ID NO:78/ SEQ ID NO:79) or the chimeric PDS/PAT1 dsRNA (SEQ ID NO:68/SEQ ID NO:69) but not in the cells treated with the mutated trigger (SEQ ID NO:70/SEQ ID NO:71) or the trigger with blunt ends (SEQ ID NO:72/SEQ ID NO:73). The results further validate the activity of the directional triggers, indicating the importance of percentage complementarity in the target-specific sequences and the importance of the overhang length and orientation in proper processing of the siRNAs.

SEQUENCE LISTING

<110> MONSANTO TECHNOLOGY LLC
IANDOLINO, ALBERTO
SANCHEZ, JUAN PEDRO
<120> COMPOSITIONS AND METHODS FOR REGULATING GENE EXPRESSION VIA RNA INTERFERENCE
<130> P34087WO01
<150> 62/015,950
<151> 2014-06-23
<160> 82
<170> PatentIn version 3.5
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<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 18
ctatcgacgt caacatgaa 19
<210> 19
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 19
tgctatcgac gtcaacatg 19
<210> 20
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 20
caacatgaac aaaatgccag atgttgctat gac 33
<210> 21
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 21
aacaaaatgc cagatgttgc tatg 24
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 22
aatgccagat gttgctatga c 21
<210> 23
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 23
gcgtgctatc gacgtcaaca tgaaca 26
<210> 24
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 24
gtgctatcga cgtcaacatg aaca 24
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 25
gcgtgctatc gacgtcaaca tgaa 24
<210> 26
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 26
tgctatcgac gtcaacatga aca 23
<210> 27
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 27
cgtgctatcg acgtcaacat gaa 23
<210> 28
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 28
gctatcgacg tcaaca 16
<210> 29
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 29
gtgctatcga cgtcaacatg aa 22
<210> 30
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 30
ctatcgacgt caacatgaac a 21
<210> 31
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 31
tgctatcgac gtcaacatga a 21
<210> 32
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 32
aaatgccaga tgttgctatg ac 22
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 33
caaaatgcca gatgttgcta tg 22
<210> 34
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 34
cgacgtcaac atgaacaaaa tgcc 24
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 35
atcgacgtca acatgaacaa aatg 24
<210> 36
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 36
tcaacatgaa caaaatgcca gatgttgcta tg 32
<210> 37
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 37
cgtcaacatg aacaaaatgc caga 24
<210> 38
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 38
ctatcgacgt caacatgaac aaaa 24
<210> 39
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 39
tgctatcgac gtcaacatga acaa 24
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 40
gtgctatcga cgtcaacatg aa 22
<210> 41
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 41
gcgtgctatc gacgtcaaca tg 22
<210> 42
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 42
tcgacgtcaa catgaaca 18
<210> 43
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 43
tatcgacgtc aacatgaa 18
<210> 44
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 44
tgctatcgac gtcaacatga acaa 24
<210> 45
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 45
cgtgctatcg acgtcaacat gaac 24
<210> 46
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 46
caaaatgcca gatgttgcta tga 23
<210> 47
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 47
aacaaaatgc cagatgttgc tat 23
<210> 48
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 48
gctatcgacg tcaacatgaa 20
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 49
gtgctatcga cgtcaacatg 20
<210> 50
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 50
caaaatgcca gatgttgcta tg 22
<210> 51
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 51
aacaaaatgc cagatgttgc ta 22
<210> 52
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 52
ctatcgacgt caacatgaac 20
<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 53
tgctatcgac gtcaacatga 20
<210> 54
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 54
tgccagatgt tgctatgac 19
<210> 55
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 55
atgccagatg ttgctatga 19
<210> 56
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 56
aaatgccaga tgttgctatg 20
<210> 57
<211> 45
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 57
gucauggcca caucuggcau uuuguucaug uucacgucaa uggca 45
<210> 58
<211> 53
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 58
gcgcgguggc cucuggcauu uuguucaugc uuucauugaa uguuccuucc caa 53
<210> 59
<211> 54
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 59
gcgcgguggc cuuuucggcg gaguaaucac uuuuuauuuu gcccacccuu acaa 54
<210> 60
<211> 55
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 60
gcgcgguggc cuccauggcc ucgaagauac uuuuuaagac cgguuuucuu gacua 55
<210> 61
<211> 48
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 61
gucauugcua caucaggcau uuuguucaug uugacaucaa uagcccgc 48
<210> 62
<211> 53
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 62
gcgcgguggc cucuggcauu uuguucaugc uuucauugaa uguuccuucc caa 53
<210> 63
<211> 53
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 63
gcgcgguggc cucauugaau guuccuuccc uuucuggcau uuuguucaug caa 53
<210> 64
<211> 57
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 64
gcgcgguggc uuagcuuuag ccaacaaacc aguuuuugac auggcaauaa acaccuc 57
<210> 65
<211> 124
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polynucleotide
<400> 65
<210> 66
<211> 50
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 66
gagccugguc uucuugcuaa aaaggugaug uuaaugggua caaaggccac 50
<210> 67
<211> 57
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 67
gcgcgguggc cuuuguaccc auuaacauca ccuuuuuagc aagaagacca ggcucgc 57
<210> 68
<211> 51
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 68
ggugguuuau ugccauguca aaaaugcaac gauuuggaau gaaaaggcca c 51
<210> 69
<211> 57
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 69
gcgcgguggc cuuuucauuc caaaucguug cauuuuugac auggcaauaa acaccuc 57
<210> 70
<211> 106
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polynucleotide
<400> 70
<210> 71
<211> 106
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic polynucleotide
<400> 71
<210> 72
<211> 57
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 72
gagguguuua uugccauguc aaaaaugcaa cgauuuggaa ugaaaaggcc accgcgc 57
<210> 73
<211> 57
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 73
gcgcgguggc cuuuucauuc caaaucguug cauuuuugac auggcaauaa acaccuc 57
<210> 74
<211> 50
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 74
gcguuacguc uaagcguccg gcgguccgua gcguauaccg cggacguaac 50
<210> 75
<211> 50
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 75
guuacguccg cgguauacgc uacggaccgc cggacgcuua gacguaacgc 50
<210> 76
<211> 51
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 76
ggagauuaca caaaacagaa guacuuagcu uccauggaag gcgcuguccu u 51
<210> 77
<211> 51
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 77
ggacagcgcc uuccauggaa gcuaaguacu ucuguuuugu guaaucucca g 51
<210> 78
<211> 49
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 78
guuguuaaga uggcaaaggc acugcaacga uuuggaauga aaagagcac 49
<210> 79
<211> 49
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 79
gugcucuuuu cauuccaaau cguugcagug ccuuugccau cuuaacaac 49
<210> 80
<211> 5
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide linker
<400> 80
aaaag 5
<210> 81
<211> 59
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 81
gcgcgguggc cuuugacaug gcaauaaaca ccuuuuuagc uuuagccaac aaaccaggc 59
<210> 82
<211> 99
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide
<400> 82

Claims

A double stranded RNA (dsRNA) molecule comprising:
a. a first strand comprising in the 5' to 3' direction:
i. a first sequence that is identical to at least 18 consecutive nucleotides of a first target nucleotide sequence; and

ii. a second sequence that is identical to at least 18 consecutive nucleotides of a second target nucleotide sequence; and

b. a second strand comprising in the 5' to 3' direction, a 5'-overhang, a nucleotide sequence that is complementary to the first strand, and a 2 nucleotide 3'-overhang, wherein the 5'-overhang is at least 5 nucleotides in length.
A double stranded RNA (dsRNA) molecule comprising:
a. a first strand comprising in the 5' to 3' direction,
i. a first nucleotide sequence that is identical to at least 18 consecutive nucleotides of a first target nucleotide sequence;

ii. a second nucleotide sequence comprising 2 or more As; and

iii. a third nucleotide sequence that is identical to at least 18 consecutive nucleotides of a second targeted nucleotide sequence or at least 18 consecutive nucleotides of the first target nucleotide sequence; and

b. a second strand comprising in the 5' to 3' direction, a 5 nucleotide 5'-overhang, a nucleotide sequence that is complementary to the first strand, and a 2 nucleotide 3'-overhang.
The dsRNA molecule of claim 1, wherein
a) the 5'-overhang of the second strand has a high GC content;

b) the 5'-overhang of the second strand is 5 nucleotides in length;

c) the 5 nucleotide 5'-overhang of the second strand has the sequence GCGCG;

d) the 2 nucleotide 3'-overhang of the second strand has the sequence UA;

e) the first strand further comprises the nucleotides GCCAC located 3' to the nucleotide sequence that is identical to at least 18 consecutive nucleotides of the target nucleotide sequence;

f) the 3' end of the first strand has a high GC content;

g) the 3' end of the first strand is not identical to the target nucleotide sequence; or

h) any combination thereof.
The dsRNA molecule of claim 2, wherein
a) the 5'-overhang of the second strand has a high GC content;

b) the 5 nucleotide 5'-overhang of the second strand has the sequence GCGCG;

c) the 2 nucleotide 3'-overhang of the second strand has the sequence UC;

d) the first strand further comprises the nucleotides GCCAC located 3' to the third nucleotide sequence; or

e) any combination thereof.
The dsRNA molecule of claim 1or 2, wherein the target nucleotide sequence is a coding region of a mRNA, a 5' untranslated region, a 3' untranslated region, an intron, a promoter, an enhancer, a terminator, an rRNA, a tRNA, a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a non-coding RNA involved in RNA interference, and any combination thereof.
A composition comprising the dsRNA molecule of any one of claims 1 to 5.
A method of regulating expression of at least one target gene, comprising applying onto the surface of a plant or plant part the composition of claim 6, wherein the dsRNA molecule comprises a first strand comprising a nucleotide sequence that is identical to at least 18 consecutive nucleotides of the target gene.
The method of claim 7, wherein the first strand of the dsRNA molecule comprises a first nucleotide sequence that is identical to at least 18 consecutive nucleotides of a first target gene and a second nucleotide sequence that is essentially identical to at least 18 consecutive nucleotides of a second target gene.
The method of claim 8, wherein the first strand of the dsRNA molecule further comprises a third nucleotide sequence that is identical to at least 18 consecutive nucleotides of a third target gene.
The method of claim 9, wherein the first strand of the dsRNA molecule further comprises a fourth nucleotide sequence that is identical to at least 18 consecutive nucleotides of a fourth target gene.
The method of claim 7, wherein the dsRNA molecule transfers from the surface of the plant or plant part into a cell of the plant or plant part.
A plant, plant part, or seed comprising the dsRNA molecule of any one of claims 1 to 5, wherein the dsRNA molecule is exogenous to the plant, plant part, or seed.
The plant, plant part, or seed of claim 12, wherein the dsRNA molecule suppresses the expression of at least one, at least two, at least three, or at least four target genes in the plant, plant part, or seed.
A method of improving the efficiency of a dsRNA molecule in producing desired small RNAs in a plant or plant part, comprising providing to the plant or plant part a dsRNA molecule of any one of claims 1 to 5, wherein the production of the 21-24 nucleotide small RNAs is directionally biased towards the 3' end of the second strand of the dsRNA molecule.